Enterocin 55 produced by non rabbit-derived strain Enterococcus faecium EF55 in relation with microbiota and selected parameters in broiler rabbits

International Journal of Environmental & Agriculture Research (IJOEAR) ISSN:[2454-1850] [Vol-3, Issue-2, February- 2017]
Page | 45
Enterocin 55 produced by non rabbit-derived strain
Enterococcus faecium EF55 in relation with microbiota and
selected parameters in broiler rabbits
Andrea Lauková1
, Ľubica Chrastinová2
, Iveta Plachá3
, Renáta Szabóová4
, Anna
Kandričáková5
, Monika Pogány Simonová6
, Zuzana Formelová7
, Ľubomír Ondruška8
,
Mária Goldová9
, Mária Chrenková10
, Viola Strompfová11
1,3,5,6,11
Institute of Animal Physiology Slovak Academy of Sciences, Košice
2,7,8,10
National Agricultural and Food Centre, Nitra-Luţianky, Slovakia
4,9
University of Veterinary Medicine and Pharmacy, Košice, Slovakia
Abstract— Ent55 is produced by poultry strain Enterococcus faecium EF55. It is substance which can be allotted to Class II
enterocins; thermo-stable, small peptide. Because producer strain has shown beneficial effect in poultry and broiler rabbits as
well, we decided to apply Ent55 in broiler rabbit husbandry. Ent55 showed antimicrobial activity in broiler rabbits by
reduction of staphylococci, Clostridiae, pseudomonads and coliforms. Its beneficial effect was demonstrated by stimulation
of phagocytic activity as well as by reduction of Eimeria spp. oocysts. GPx values were lower; it means, no oxidative stress
was evoked. Moreover, it has not negative influence on growth performance and biochemical parameters. Our results
indicated that enterocin produced by not-autochtonous strain can also have protective and beneficial effect in broiler rabbits.
Keywords— Enterocin, effect, microbiota, immunity, rabbit.
I. INTRODUCTION
Ent55 is produced by poultry strain Enterococcus faecium EF55 (1, 2). It is substance which can be allotted to Class II
enterocins; thermo-stable, small peptide. Its maximum inhibition activity achieved after 12 h of cultivation in broth was
12 800 AU/ml. Ent55 has a broad antimicrobial spectrum and its inhibition activity in vivo was demonstrated in Japanese
quails; but its producer strain was shown to have also benefits in chicken. That is, Levkut et al. (3) reported inhibition
activity of EF55 strain against Salmonella Enteritidis PT4 in chicks. Administration of EF55 strain to chicks challenged with
Salmonella Enteritidis (SE147) resulted in increased number of blood heterophils, caecal IgA+ IEL (4). This strain also
demonstrated positive impact on intestinal morphometry in the jejunum (5). To have this information we decided to test
Ent55 effect in broiler rabbits. The aim was to spread information concerning the Ent55 but also to find beneficial activity in
rabbits by this non rabbit-derived strain producing Ent55. In last decade, bacteriocins (enterocins) have been tested as
biopreservative substances in food industry to prevent contamination by spoilage bacteria (6, 7). However, at recent time,
more frequently have been presented studies with application of bacteriocins to protect animals health including our studies
(8, 9, 10, 11) .
II. MATERIAL AND METHODS
2.1 Experiment design
Forty-eight post-weaned rabbits (aged 5 weeks, Hyplus breed) both sexes were divided into the experimental group (EG) and
the control group (CG); 24 animals in each group. Animals were kept in the standard cages (0.61m x 0.34 cm x 0.33 m, the
type D-KV-72 supplied by Kovobel, Czech Republic), two animals per cage. The cages allowed faeces separation. A cycle of
16 h of light and 8 h of dark was used throughout the experiment. The temperature and humidity were recorded continuously
with a digital thermograph positioned at the same level as the cages. The heating and forced ventilation systems allowed the
building air temperature to be maintained within 16 ± 4ºC through the experiment. The relative humidity was about 70 ± 5 %.
All care and experimental procedures were approved by the Slovak Veterinary and Food Administration. The experiment
was performed in co-operation with our colleagues in Nitra-Luţianky (National Agricultural and Food Centre, Slovakia). The
rabbits were fed the commercial feed mixture for broiler rabbits (pelets 3.5 mm in average size) characterized by the contain
of 901.75 g/kg dry matter, 828.29 g/kg of organic matter, 176.02 g/kg of fibre content, 155.89 g/kg of N-substances, 34.65
g/kg of fat content, 73.46 g/kg of ash content; digested energy was 11.01 MJ/kg and metabolised energy 10.46 MJ/kg.
Samples of individual feeds and complete granulated mixture were analyzed according to STN 46 7092 (Slovak Technical
Norm, 2010). The rabbits had access to feed and water ad libitum. Water and feed consumption by the rabbits were

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controlled daily. Rabbits in EG were administered enterocin 55 (semi-purified) prepared as previously reported Strompfová
and Lauková (2). Its activity was checked by agar diffusion method (13) against the indicator strain Enterococcus avium EA5
(our strain). Ent55 was applied in the water at a dose 50 µl per animal per day for 21 days (3 weeks). The experiment
duration was 42 days.
2.2 Sampling and microbial analyses
Faecal sampling was carried out on day 0-1 (the start of experiment; 10 mixture samples from 48 rabbits – initial microbial
background), on day 21 (3 weeks of enterocin 55-Ent55) application; five mixture samples from each group) and on day 42;
the end of experiment, 3 weeks after Ent 55 cessation; five mixture samples from each group). The microbial counts were
checked to follow antimicrobial effect of Ent55 using the standard microbiological method (ISO, International Organization
for Standardization): the appropriate dilutions of samples in Ringer solution (pH 7.0; Oxoid Ltd., Basingstoke, Hampshire,
United Kingdom) were plated onto media according to ISO: M-Enterococcus agar for enterococci (ISO-7889, Difco, Detroit,
USA), Baird-Parker agar supplemented with egg yolk tellurite solution (ISO 21527-1, Becton and Dickinson, Cockeysville,
USA) for isolation of coagulase-positive staphylococci (CoPs), Mannitol Salt agar, Clostridium difficile agar with the
supplement SR0096E and 7% (v/v) defibrinated horse blood (SR0050, ISO 15883, Oxoid Ltd., Basingstoke, Hampshire,
United Kingdom) were used to enumerate coagulase-negative staphylococci (CoNS) and Clostridium spp. Mac Conkey agar
(Oxoid) was used to detect coliform bacteria. Pseudomonads were isolated on Cetrimide agar (ISO 16266, Becton and
Dickinson). The plates were incubated at 30 ºC and/or 37 ºC for 24-48 h depending on the bacterial genera. The bacterial
counts were expressed in colony-forming units (log10) CFU per gram ± SD. On day 21 and 42, three animals (n=3) from
each group were slaughtered. They were electrically stunned and killed by cutting the carotidis and jugular veins (14). One g
of caecal content and appendix was treated according to the standard microbiological dilution method mentioned above.
Caecal and appendix samples were plated on the media as already indicated.
2.3 Blood analysis including phagocytic activity and enzyme gluthation-peroxidase
Blood (n=6) was sampled from the marginal ear vein (vena auricularis) on day 0-1, mixed blood samples, and on day 21, 42
from each group. Phagocytic activity (PA) was measured using the direct counting procedure with microspheric hydrophilic
particles (MSHP). Ingestion of MSH particles by polymorphonuclear cells (PMNs) was determined with a modified test (15):
50 ml of MSH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100 ml of blood in an Eppendorf-type
test tube and incubated at 37°C for 1 h. Blood smears were then prepared and stained by May-Grünwald and Giemsa-
Romanowski (Merck, Germany). In each smear, 100 cells were observed to determine the relative number of white cells
containing at least three engulfed particles (phagocytic activity), and the index of phagocytic activity (IPA, number of
engulfed particles/total number of neutrophils and monocytes observed). The percentage of phagocytic cells was evaluated
using an optical microscope (Motic BA 400 Biological Microscope- Motic China Group Co., Ltd.) by counting PMN up to
100.
The activity of glutathione-peroxidase (GPx; U/gHb) in blood (n=6) was determined using a specific commercial kit
(RANSEL, Randox, United Kingdom) according to the spetrophotometric assay procedure of Paglia and Valentine (16).
Total proteins (g/l), albumine (g/l), triglycerides, cholesterol, calcium (mmol/l), glucose (g/l), alanine-aminotranspherase
(µkat/l) were performed using commercial kits from Randox (United Kingdom). Blood (n=6, from each group) was sampled
into dry non-heparinized Ependorf tubes; blood serum was separated by centrifugation at 3000 x g for 10 min, then stored
frozen in plastic vials until analysis.
2.4 Eimeria oocysts counting, acids in caecal content, growth performance
To detect Eimeria sp. oocysts the quantitative MacMaster method was used (1968); oocysts counts were expressed in OPG/g
(detected oocysts per gram of faecal sample). Lactic acid and organic acids (acetic, propionic and butyric) were evaluated
using gas chromatography (Perkin Elmer, USA). Weight mass, feed conversion amd mortality were checked, measured and
mathematically calculated.
Statistical evaluation of the results was performed using one-way ANOVA test with Tukeys post hoc test (the level of
significance set at p<0.05 ± standard deviation - SD).

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III. RESULTS
Antimicrobial activity of Ent55 in faeces was demonstrated by significant reduction of CoNS in EG on day 21 compared to
EG on day 42 (2. sampling to 3. sampling, a:b, p<0.001, Table 1). Pseudomonas spp. in EG were reduced in 2. sampling-day
21 compared to day 0-1 (1. sampling, a:b, a:c, p<0.05). Coliforms were reduced in EG on day 42 (sampling 3.) compared to
day 0-1 (1. sampling, a:c, p<0.05; d:c p<0.05). On day 21 coliforms were reduced significantly in EG compared to CG (d:e,
p<0.05) with reductive tendency on day 42 (c:f, p=0.054).
TABLE 1
PROFILE OF BACTERIA IN FACES OF BROILER RABBITS DURING THE EXPERIMENT (LOG10 CFU/G) ± SD
Sampling EG CG
(1) Day 0-1 (n=8)
Enterococci 3.20 (0.36) 3.20 (0.36)
LAB 3.02 (0.46) 3.02 (0.46)
CoNS 3.50 (0.10)a
3.50 (0.10)
CoPS 2.20 (0.23) 2.20 (0.23)
Cl. spp. 2.71 (0.40) 2.71 (0.40)
Ps.spp. 5.43 (0.90)a
5.43 (0.90)
Coliforms 4.50 (1.63)a
4.50 (1.63)
(2) Day 21 (n=5)
Enterococci 3.47 (0.23) 3.56 (0.83)
LAB 3.21 (0.70) 2.83 (0.77)
CoNS 3.30 (0.15)b
3.06 (0.58)
CoPS 2.43 (0.24) 2.24 (0.09)
Cl. spp. 4.54 (0.82) 4.63 (0.90)
Ps.spp. 4.18(0.30)b
4.44 (0.62)
Coliforms 2.95 (0.65)d
3.95 (0.37)e
(3) Day 42 (n=5)
Enterococci 3.85 (0.69) 2.68 (1.11)
LAB 3.28 (0.89) 2.23 (1.32)
CoNS 4.04 (0.08) 3.96 (0.21)
CoPS 2.19 (0.92) 2.14 (0.53)
Cl. spp. 4.81 (1.44) 5.80 (0.90)
Ps.spp. 4.32 (0.88) 4.89 (0.12)
Coliforms 1.53 (0.84)c
3.23 (1.46)f
EG-experimental group, CG-control group, Day 0-1, start of experiment, Day 21, after 3 weeks of application, Day 42, end
of experiment-3 weeks after cessation, LAB-lactic acid bacteria, CoNS-coagulase-negative staphylococci, CoPS, coagulase-
positive staphylococci, Cl. Spp.-Clostridium spp.; Ps spp.-Pseudomonas spp., Coliform bacteria; Significant reduction in
CoNS of 1. sampling compared to 2. sampling (a:b, p<0.05); Pseudomonas spp. were lower in 1. sampling compared to 2.
sampling (a:b, p<0.05), Coliforms were lower in 3. sampling compared to 1. sampling (a:c, p<0.01); On day 21, coliforms
were significantly reduced in EG compared to CG (d:e, p<0.05) with reduction tendency on day 42 (c:f, p=0.054);
Microbial counts in caecum and appendix were lower than in faeces of broiler rabbits. Similarly as in faeces, the counts of
CoNS were reduced in EG on day 21 (2. sampling) compared to day 42 (3. sampling, a:b, p<0.01, Table 2). On day 21,
Pseudomonas spp. were lower in EG compared to CG (b:b, difference 0.64 log cycle). Coliforms were also lower in EG
compared to CG on day 21 (c:c, difference 1.64 log cycle). On day 42, Clostridium spp. in EG and coliforms were lower
compared to CG with difference 0.46 log cycle (d:d) and 1.66 log cycle (e:e).

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TABLE 2
PROFILE OF BACTERIA IN CAECUM OF BROILER RABBITS DURING THE EXPERIMENT (log10 CFU/g) ± SD
Sampling EG CG
(2) Day 21 (n=3)
Enterococci 1.09 (0.33) 1.36 (0.63)
LAB 1.37 (0.72) 1.23 (0.41)
CoNS 3.49 (0.17)a
3.77 (0.08)
CoPS 2.27 (0.29) 1.94 (0.42)
Cl. spp. 5.10 (0.00) 5.10 (0.00)
Ps.spp. 3.71(0.23)b
4.35 (0.43)b
Coliforms 1.94 (1.50) 3.58 (1.34) c
(3) Day 42 (n=3)
Enterococci 3.85 (0.69) 1.54 (0.60)
LAB 3.28 (0.89) 1.27 (0.35)
CoNS 4.04 (0.08)b
3.87 (0.29)
CoPS 2.19 (0.92) 1.95 (0.42)
Cl. spp. 4.81 (1.44)d
5.27 (0.29d
Ps.spp. 4.32 (0.88) 4.44 (0.51)
Coliforms 1.53 (0.84)e
3.19 (1.98e
EG-experimental group, CG-control group, Day 21, after 3 weeks of application, Day 42, end of experiment-3 weeks after
cessation, LAB-lactic acid bacteria, CoNS-coagulase-negative staphylococci, CoPS, coagulase-positive staphylococci, Cl.
Spp.-Clostridium spp.; Ps spp.-Pseudomonas spp., Coliform bacteria; On day 21 (2. sampling) CoNS in EG were lower than
at day 42 (3. sampling (a:b, p<0.01); at day 21, Pseudomonas spp. were lower in EG compared to CG (b:b, difference 0.64
log cycle); Coliforms in EG were lower than in CG (c:c, differemce 1.64 log cycle); On day 42, in EG Clostridium spp., and
coliforms were lower than in CG with differences 0.46 log cycle (d:d), 1.66 log cycle (e:e);
Coliforms were reduced also significantly in appendix of EG on day 21 compared to day 42 (a:b, p<0.05, Table 3). On day
42, Clostridium spp., Pseudomonas spp. and coliforms were lower in EG compared to CG with differences 0.87 log cycle
(c:c), 1.07 log cycle (d:d) and 1.11 log cycle (b:e). CoPS, enterococci and LAB were neither influenced by Ent55 in faeces,
nor in caecum and appendix.
TABLE 3
PROFILE OF BACTERIA IN APPENDIX OF BROILER RABBITS DURING THE EXPERIMENT (log10 CFU/g) ± SD
Sampling EG CG
(2) Day 21 (n=3)
Enterococci 3.00 (0.12) 1.47 (0.53)
LAB 1.90 (1.07) 2.33 (1.24)
CoNS 2.78 (0.48) 2.34 (0.25)
CoPS 2.60 (0.71) 1.57 (0.58)
Cl. spp. 5.06 (0.03) 5.04 (0.00)
Ps.spp. 4.60(0.87) 5.10 (0.00)
Coliforms 2.28 (1.21)a
5.10 (0.00)
(3) Day 42 (n=3)
Enterococci 1.74 (1.00) 1.63 (0.50)
LAB 1.10 (0.20) 0.90 (0.00)
CoNS 3.50 (0.17) 3.33 (0.25)
CoPS 1.62 (0.13) 1.83 (0.33)
Cl. spp. 4.23 (1.03)c
5.10 (0.00)c
Ps.spp. 4.11 (0.73)d
5.18 (1.48)d
Coliforms 3.68 (2.60)b
4.79 (2.16)
cessation, LAB-lactic acid bacteria, CoNS-coagulase-negative staphylococci, CoPS, coagulase-positive staphylococci, Cl.
Spp.-Clostridium spp.; Ps. spp.-Pseudomonas spp., Coliform bacteria; reduction of coliforms in EG on day 21 compared to
EG on day 42 (a :b, p<0.05), On day 42, reduced counts of Clostridium spp., Pseudomonas spp. and coliforms in EG
compared to CG with differences 0.87 log cycle (c:c), 1.07 log cycle (d:d), 1.11 log cycle (b:e).

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On day 21, phagocytic activity was stimulated in EG compared to CG by significant increase (a:a, p<0.01) as well as on day
42 (b:b, p<0.001, Table 4). The values of GPx were as follows: on day 21 in EG-146.70 (22.93) compared to CG-144.14
(20.73 U/g Hb). On day 42 they were decreased in both, EG-128.08 (34.94) U/g Hb; 124.76 (30.78) IN CG.
TABLE 4
PHAGOCYTIC ACTIVITY IN EXPERIMENTAL AND CONTROL RABBITS AND INDEX OF PHAGOCYTIC ACTIVITY
(%) ± SD
EG CG
PA21 49.00 (2.37)a
43.83 (2.40)a
PA42 50.50 (1.05)b
43.33 (2.50)b
IPA21 2.35 (0.15) 2.33 (0.14)
IPA42 2.20 (0.18) 2.13 (0.23)
On day 0-1, PA was 43.00 (1.67)% and IPA 2.30 (0.17); On day 21, EG:CG, a:a, p<0.01; On day 42, EG:CG, b:b, p<0.001;
EG-experimental group-Ent55 application, CG-control group; PA21- phagocytic activity on day 21 (3 weeks application);
PA42-phagocytic activity on day 42 (3 weeks cessation); IPA-index of phagocytic activity;
Biochemical parameters, such as total proteins, albumine, triglycerides, cholesterol, glucose, ALT and Ca were influenced in
the range of reference values; wether higher, they were decreased or increased wether low (Table 5). On day 21 and 42,
decrease of Eimeria oocysts was noted in EG rabbits compared to CG (Table 6). EG samples compared to CG were oocysts
absent; in EG oocysts were not counted. On day 21, acids values in caecal contents were slightly increased (Table 7).
TABLE 5
BIOCHEMICAL PARAMETERS IN BLOOD SERUM
n=6 EG CG
TP (g/l)
Day 0-1 48.5 (2.9) 48.5 (2.9)
Day 21 52.9 (5.0) 53.2 (1.9)
Day 42 57.3 (2.5) 56.3 (3.3)
ALB (g/l)
Day 0-1 41.8 (5.2) 41.8 (5.2)
Day 21 45.0 (3.7) 42.6 (1.4)
Day 42 44.7 (2.9) 46.9 (2.5)
TRIG (mmol/l)
Day 0-1 2.13 (0.33) 2.13 (0.33)
Day 21 2.00 (0.50) 1.74 (0.19)
Day 42 1.83 (0.13) 1.90 (0.31)
CHOL (mmol/l)
Day 0-1 3.57 (0.54) 3.57 (0.54)
Day 21 3.26 (0.41) 3.02 (0.21)
Day 42 2.75 (0.26) 2.83 (0.27)
GLU (g/l)
Day 0-1 7.69 (0.37) 7.69 (0.37)
Day 21 8.52 (1.08) 8.60 (1.16)
Day 42 7.69 (0.39) 7.44 (0.56)
ALT (µkat/l)
Day 0-1 0.05 (0.02) 0.05 (0.01)
Day 21 0.11 (0.04) 0.10 (0.02)
Day 42 0.09 (0.03) 0.12 (0.04)
Ca (mmol/l)
Day 0-1 2.89 (0.10) 2.89 (0.10)
Day 21 3.07 (0.09) 3.15 (0.19)
Day 42 3.15 (0.15) 3.12 (0.09)

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TABLE 6
EIMERIA OOCYSTS IN OOCYST PER GRAM OF FAECES (OPG/g)
Sampling EG CG
Day 21 (n=5) neg 1760 (41.95)
Day 42 (n=5) neg 600 (28.63)
cessation
TABLE 7
LACTIC ACID AND OTHER ACIDS IN CAECAL CONTENT OF BROILER RABBITS
EG CG
Day 21
LA (g/100g) 0.084 0.097
AA (mmol/l) 10.908 7.637
PA (mmol/l) 0.615 0.345
BA (mmol/l) 1.921 1.488
Day 42
LA (g/100g) 0.102 0.106
AA (mmol/l) 7.628 7.201
PA (mmol/l) 0.377 0.399
BA (mmol/l) 1.634 1.451
cessation; LA-lactic acid, AA-acetic acid , PA-propionic acid, BA-butyric acid
An average live weight mass at the end of experiment in rabbits of EG reached 2796.33 (152.47) g. In CG it was slightly
less-2624.76 (68.24) g. It was reached from the initial values of 985.00 (105.82)g for CG and 984.17 (113.75) g for EG. No
mortality was observed. Regarding the feed conversion in the period of 3 weeks, it was 2.91 g per g live weight mass in CG
and in EG it was 2.45 g/g of live weight mass.
IV. DISCUSSION
Bacteriocins were identified almost a century ago; it is believed that 99% of bacteria produce at least one bacteriocin (18).
The kingdom of bacteriocins is rich and contains a diversity of small proteins from Gram-positive and Gram-negative
bacteria. Several bacteria produced by lactic acid producing Firmicutes offer potential application (18). Bacteriocins are even
anticipated to be good candidates for therapeutic application in order to fight against pathogenic bacteria (19). The genus
Enterococcus belongs to Firmicutes and a large number of bacteriocins produced by enterococci (mostly enterocins) have
been described and characterized at the biochemical and genetic level (20, 21, 22, 23, 24, 25). Enterocins are known to have
frequently a broad antimicrobial spectrum. Many in vitro studies were related to this statement (20, 24, 22, 23). However,
concerning the application abilities in animals to protect husbandry has been reported only limitedly. Our working group has
published several beneficial effects reached by different enterocins produced by different strains in especially food-producing
animals such as broiler rabbits, poultry but also in horses (8, 9). Previously, bacteriocins were characterized that they act
against closely related bacteria (26) but many studies have spread last decade this statment because e.g. enterocins can inhibit
more or less related bacteria, spoilage bacteria including (27). Ent55 also showed antimicrobial inhibition effect against
Gram-positive CoNS and Clostridium spp.; however, coliform bacteria and Pseudomonads were also inhibited by significant
reduction or by bacteriostatic effect on their growth. Similarly as Ent55, our other enterocins confirmed this inhibition; it is
e.g. Ent M (produced by probiotic strain E. faecium AL41-CCM8558) reduced staphylococci, coliforms, Pseudomonas spp.
and Clostridiae (28) or firstly described Ent4231 produced by E. faecium CCM4231- animal/ruminal origin strain also
showed antimicrobial activity (8). In spite of the fact that amino sequence analysis of Ent55 is not tested, their properties are
more similar to Class II enterocins. Moreover, producer strain of Ent55 (E. faecium EF55) was very active in chicken (1).
Interesting is inhibition of bacteria in appendix. Appendix has importance from the aspect of differentiation and forming of
lymphoid cells which after migration in the secondary lymphatic organs (spleen, lymphatic nodes and lymphoid tissue of the
digestive tract-GALT) are multiplied (29). It shows that appendix can play role in eliminating and killing of undesirable
microbiota performing by this protection of organism.

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Phagocytic activity is important parameter; there polymorphonuclear leucocytes are responsible for non-specific immune
response and in first line share of phagocytosis introdefence of the host to infectious and inflammatory actions (30). This
non-specific immunity has been positively-increasingly influenced in our previous studies by using the other enterocins such
as Ent4231 or Ent2019 or EntM (8, 9, 10). We could suppose that rabbits adopt to substance because prolonged stimulative
effect on PA was measured. This stimulative and protective effect of enterocins could be associated with increase of PA.
Moreover, oxidative stress was not evoked by additive. In the case of EntM-producing strain AL41 in poultry even lower
counts of GPx were measured in EG compared to CG (31). Very important and original is finding that Eimeria oocysts were
reduced. We dont know exact mode of action; however, it could be explained probably through the immunity stimulation; it
means higher immunity, less sensitivity to agens as Eimeria also are. Pogány Simonová et al. (32) Szabóová et al. (8) also
reported reduction of Eimeria oocysts in case of Ent2019 or/and Ent4231; it was studied at first in association with Eimeria
oocyst reduction in rabbits. Caecal acids were increased only in case of acetic acid. Biochemical parameters reported were in
accordance with those reported by Lauková et al. (9) after EntM application with any significant impact. Effect of enterocins
on growth performance is varied from Ent to Ent used. However, it seems that this parameter is more influenced by Ent-
producing strains than by enterocins themselves.
V. CONCLUSION
Ent55 showed antimicrobial activity in broiler rabbits by reduction of staphylococci, Clostridiae, pseudomonads and
coliforms. Its beneficial effect was demonstrated by stimulation of phagocytic activity as well as by reduction of Eimeria
spp. oocysts. GPx values were lower; so no oxidative stress was evoked. Moreover, it has not negative influence on growth
performance and biochemical parameters. Our results indicated that also enterocins produced by not-autochtonous strain can
have protective and beneficial effect in broiler rabbits.
ACKNOWLEDGEMENTS
Results achieved were financially covered by the projects Vega 2/0004/14 and Vega 2/0006/17. We are thankful Mrs.
Margita Bodnárová for her skillful laboratory work. We also thank to slaughterhouse people for their help. Part of results
concerning the microbiota and some paramaters were included in Proceedings of lectures ―XVII Workshop-New ways in
rabbits husbandry –Nové smery v intenzívních a zájmových chovech králiku (in Slovak), 6.10. Prague, 2013, p. 28-36.
REFERENCES
[1] V. Strompfová, A. Lauková and D. Mudroňová, ―Effect of bacteriocin-like substance produced by Enterococcus faecium EF55 on the
composition of avian gastrointestinal microflora‖ in Acta Vet. Brno, vol. 72, 2003, pp. 559-564.
[2] V. Strompfová and A. Lauková, ―In vitro study on bacteriocin production of Enterococci associated with chickens‖ in Anaerobe, vol.
13, 2007, pp. 228-237.
[3] M. Levkut, J. Pistl, A.Lauková, V. Revajová, R. Herich, Z. Ševčíková, V. Strompfová, R. Szabóová and T. Kokinčáková,
―Antimicrobial activity of Enterococcus faecium EF55 against Salmonella Enteritidis in chicks‖, in Acta Vet. Hungarica, vol.57,
2009, pp. 13-24.
[4] M. Levkut Jr., V. Revajová, V. Karaffová, A. Lauková, R. Herich, V. Strompfová, Z. Ševčíková, R. Ţitňan, M. Levkutová and M.
Levkut Sr., ―Evaluation of mucin and cytokines expression with intraepithelial lymphocytes determination in the caecum of broilers
administered with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis SE147‖, in J. Vet. Med. Anim. Health, vol.
8, 2016, pp. 214-222.
[5] Z. Ševčíková, J. Blanár, A. Lauková, V. Revajová, V. Strompfová, M. Levkut, ―Effect of Enterococcus faecium EF55 on
morphometry and proliferative activity of intestinal mucosa in broilers infected with Salmonella Enteritidis‖, in J. Vet. Res., vol.60,
2016, pp. 261-265.
[6] A. Lauková, S. Czikková, S. Laczková and P. Turek, ―Use of enterocin CCM4231 to control Listeria monocytogenes in
experimentally contaminated fermented Hornád salami‖, in Int. J. Food Microbiol., vol. 52, 1999, pp. 115-119.
[7] H. Chen and D.G. Hoover, ―Bacteriocins and their food applications‖, in Comp. Rev. Food Sci. Food Safety, vol. 2, 2003, pp. 82-100.
[8] R. Szabóová, A. Lauková, Ľ. Chrastinová, V. Strompfová, M. Pogány Simonová, Z. Vasilková, K. Čobanová, I. Plachá and M.
Chrenková, ―Effect of combined administration of enterocin 4231 and sage in rabbits‖, in Polish J. Vet. Sci., vol. 14, 2011, pp. 359-
366.
[9] A. Lauková, Ľ. Chrastinová, M. Pogány Simonová, V. Strompfová, I. Plachá, K. Čobanová, Z. Formelová, M. Chrenková and Ľ.
Ondruška, ―Enterococcus faecium AL41:Its Enterocin M and their beneficial use in rabbits husbandry‖, Probiotics Antimicro. Prot.,
vol. 4, 2012, pp. 243-249.
[10] M. Pogány Simonová, A. Lauková, I. Plachá, K. Čobanová, V. Strompfová, R. Szabóová and Ľ. Chrastinová, ―Can enterocins affect
phagocytosis and glutathione-peroxidase in rabbits?‖ in Cent. Eur. J. Biol., vol. 8, 2013, pp. 730-734.
[11] G. Telléz, A. Lauková, J.D. Latorre, X. Hernandez-Velasco, B.M. Hargis and T. Callaway, ―Food-producing animals and their health
in relation to human health‖, in Microbial Ecol. Health Dis., vol. 26, pp. 25876.

Page | 52
[12] Slovak Technical Norm, 2010.
[13] L. De Vuyst, R. Callewaert and B. Pot, ―Characterization of the antagonistic activity of Lactobacillus amylovorus DCE471 and large
scale isolation of its bacteriocin amylovorin L471‖ in Syst. Appl. Microbiol., vol. 19, 1996, pp. 9-20.
[14] Ľ. Chrastinová, K. Čobanová, M. Chrenková, M. Poláčiková, Z. Formelová, A. Lauková, Ľ. Ondruška, M. Pogány Simonová, V.
Strompfová, Z. Mlyneková, A. Kalafová and Ľ. Grešáková, ―Effect of dietary zinc supplementation on nutrients digestibility and
fermentation characteristics of caecal content in physiological experiment with young rabbits‖ in Slovak J. Anim. Sci. Vol. 49, 2016,
pp. 23-31.
[15] V. Vetvička, L. Fornousek., J. Kopeček, J. Kaminková, L. Kašpárek and M. Vránová, ―Phagocytosis of human blood leukocytes, a
simple micro-method‖ in Immunol. Lett., vol. 5, 1982, pp. 97-100.
[16] D.E. Paglia and W.N. Valentine, ―Studies on quantitative and qualitative characterization of erythrocyte gluthatione peroxidase‖ in J.
Lab.Cl. Med., vol. 70, 1967, pp. 158-169.
[17] Mac Master, ―Modified MacMaster egg counting for quantification of nematode eggs‖ in Ministry of Agriculture and Fishing,
London, 1986.
[18] Y. Belguesmia, A. Madi, D. Sperandio, A. Merieau, M. Feuilloley, H. Prevost, D. Drider and N. Connil, ―Growing insights into the
safety of bacteriocins:the case of enterocin S37‖ in Res. Microbiol., vol. 162, 2011, pp. 159–163..
[19] A. Parisien, B. Allain, J. Zhang, R. Mandeville and C.Q. Lan, ‖Novel alternatives to antibiotics:bacteriophages, bacterial cell wall
hydrolases, and antimicrobial peptides‖ in J. Appl. Microbiol., vol. 104, 2008, pp. 1-13.
[20] A. Lauková, M. Mareková and P. Javorský, ―Detection and antimicrobial spectrum of a bacteriocin-like substance produced by
Enterococcus faecium CCM4231‖, in Lett. Appl. Microbiol., vol. 16, 1993, pp. 257-260.
[21] L.M. Cintas, P. Casaus, H. Holo, P.E. Hernandez, I.F. Nes and L.S. Havarstein, ― Enterocins L50A and L50B, two novel bacteriocins
from Enterococcus faecium L50 are related to staphylococcal hemolysins‖ in J. Bacteriol., vol. 180, 1998, pp. 1988-1994.
[22] M. Mareková, A. Lauková, L. De Vuyst, M. Skaugen and I.F. Nes, ―Partial characterization of bacteriocins produced by
environmental strain Enterococcus faecium EK13‖ in J. Appl. Microbiol., vol. 94, 2003, pp. 523-530.
[23] M. Mareková, A. Lauková, M. Skaugen and I.F. Nes, ―Isolation and characterization of a new bacteriocin, termed enterocin M,
produced by environmental isolate Enterococcus faecium AL41‖ in J. Ind. Microbiol. Biotechnol., vol. 34, 2007, pp. 533-537.
[24] CH.M.A. Franz, M.J. van Belkum, W.H. Holzapfel and A. Gálvéz, ―Diversity of enterococcal bacteriocins and their grouping in a
new classification scheme‖ in FEMS Microbiol. Rev., vol. 31, 2007, pp. 293-310.
[25] N. Sawa, P. Wilaipun, S. Kinoshita, T. Zendo, V. Leelawatcharamas, J. Nakayama and K. Sonomoto, ―Isolation and characterization
of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1‖ in Appl. Environ. Microbiol., vol. 78,
2012, pp. 900-903.
[26] I.F. Nes and H. Holo, ―Class II antimicrobial peptides from lactic acid bacteria‖ in Biopolymers, vol. 55, 2000, pp. 50-61.
[27] I.F. Nes, H. Holo, G., Fimland, H.H. Hauge, J. Nissen-Meyer, „Unmodified peptide-bacteriocins (ClassII) produced by lactic acid
bacteria― in C.J. Dutton, M.A. Haxell, H.A.I. McArthur, R.G. Wax (Eds.), Peptide Antibiotics:Discovery, Modes of Action and
Applications, Marcel Dekker, New York/Basel, 2002.
[28] A. Lauková, M. Pogány Simonová, Ľ. Chrastinová, I. Plachá, K. Čobanová, Z. Formelová, M. Chrenková, Ľ. Ondruška and V.
Strompfová, ―Benefits of combinative application of probiotic, enterocin M-producing strain Enterococcus faecium AL41 and
Eleutherococcus senticosus in rabbits‖ in Folia Microbiol., vol. 61, 2016, pp. 169—177.
[29] L. Fortun-Lamothe and S. Boullier ― A review on the interactions between gut microflora and digestive mucosal immunity. Possible
ways to improve the health of rabbits‖ in Livest. Sci., vol. 107, 2007, pp. 1-18.
[30] B.M. Escribano, F.M. Castejón, R. Vivo, S. Aguera, E.L. Aguera and M.D. Rubido, ―Nonspecific immune response of peripheral
blood neutrophils in two horse breeds Anglo-Arabian and Spanish-Arabian:response to exercise‖ in Comp. Immunol. Microbiol.
Infect. Dis., vol. 28, 2005, pp. 145-154.
[31] A. Lauková, V. Strompfová, I. Plachá, K. Čobanová, Š. Faix and M. Pogány Simonová, ―Beneficial effect of enterocin M-producing,
probiotic strain Enterococcus faecium AL41 in model experiment with hens‖ in Global J. Anim. Sci. Res., vol. 31, 2016, pp. 206-213.
[32] M. Pogány Simonová, A. Lauková, Ľ. Chrastinová, V. Strompfová, S. Faix, Z. Vasilková, Ľ, Ľ. Ondruška, R. Jurčík and J. Rafay,
―Enterococcus faecium CCM7420, bacteriocin PPB CCM7420 and their effect in the digestive tract of rabbits ― in Czech J. Anim.
Sci., vol. 54, 2009, pp. 376-386.

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