Veterinary Parasitology 188 (2012) 3140

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Assessment of dietary supplementation with probiotics on

performance, intestinal morphology and microora of chickens
infected with Eimeria tenella
I. Giannenas a, , E. Papadopoulos b , E. Tsalie c , El. Triantallou d , S. Henikl e , K. Teichmann e ,
D. Tontis c
a
Laboratory of Animal Nutrition & Husbandry, Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece
b
Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Medicine, Aristotle University of Thessaloniki, 43100 Karditsa, Greece
c
Laboratory of Pathology, Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece
d
Military Veterinary Training and Nursing Centre, 41334 Larisa, Greece
e
BIOMIN Research Center, Technopark 1, 3430 Tulln, Austria

a r t i c l e i n f o a b s t r a c t

Article history: We evaluated the effects of dietary supplementation with different preparations of pro-
Received 20 September 2011 biotics on the performance of broiler chickens experimentally infected with 2 104
Received in revised form 13 January 2012 sporulated oocysts of Eimeria tenella at 14 days of age. Three hundred, day-old, Cobb-
Accepted 22 February 2012
500 chicks, as hatched, were separated into 10 equal groups with three replicates. Two
of the groups, one challenged with E. tenella oocysts and the other not, were given a basal
Keywords:
diet and served as controls without medication. The other challenged groups were given
Probiotics
Eimeria tenella
the anticoccidial lasalocid (60 mg/kg) or Enterococcus faecium (5 108 or 5 109 cfu/kg
Experimental infection feed), Bidobacterium animalis (5 108 cfu/kg feed), Lactobacillus reuteri (5 108 cfu/kg
Growth performance feed), Bacillus subtilis (5 108 cfu/kg feed), or a multi-species probiotic mix at 5 108 or
Chicken 5 109 cfu/kg feed, respectively. The trial lasted 6 weeks. Individual body weight, feed
intake per pen and feed conversion ratio values were recorded weekly, along with the
extent of bloody diarrhea, excreta oocyst numbers and bird mortality. Caecal lesions were
assessed and intestinal samples were taken for histopathological and bacteriological evalu-
ation from ileum and caecum. Overall growth performance of chickens fed the multi-species
probiotic mix at both levels was higher (P < 0.05) compared to the infected control. Overall
oocyst shedding was lowest (P < 0.05) in the lasalocid supplemented group. Villous height
was higher (P < 0.05) in Bacillus supplemented groups compared to infected controls. The
Lactobacillus supplemented group had the highest (P < 0.05) numbers of both Lactobacillus
and Bidobacterium in ileum and caecum. In conclusion, dietary probiotics are promising
for further investigation on improving intestinal health and growth performance of broiler
chickens experimentally challenged with E. tenella.
2012 Elsevier B.V. All rights reserved.

1. Introduction utilization and impaired growth rate in poultry industry

Avian coccidiosis is the major parasitic disease of
poultry causing mortality, malabsorption, inefcient feed
Corresponding author. Tel.: +30 6977629197; fax: +30 2441066041.
E-mail address: giannenas@vet.uth.gr (I. Giannenas).
(Lee et al., 2007). Outbreaks of disease caused by resis-
tant Eimeria strains are frequent triggering high economic
impact on production in various parts of the world (Peek,
2010).
Conventional disease control strategies rely mainly
on chemoprophylaxis, which is a tremendous cost to
the industry. Existing vaccines consist of live virulent or

0304-4017/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2012.02.017
32 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140
attenuated Eimeria strains with limited scope of protection
against an ever-evolving and widespread pathogen. The
continual emergence of drug-resistant strains of Eimeria,
coupled with the increasing regulations and bans on the use
of anticoccidial drugs in commercial poultry production,
urges the need for novel approaches and alternative control
strategies (Giannenas et al., 2003). Consumers also demand
food products of animal origin without drug residues
or pharmaceutical metabolites (Dalloul and Lillehoj,
2006).
Probiotics are live, non-pathogenic bacteria that con-
tribute to the health and balance of the intestinal tract.
Probiotics have been used in broiler chickens (Mohan et al.,
1996; Dalloul et al., 2002, 2003; Patterson et al., 1997;
Mountzouris et al., 2007), in order to assess their bene-
cial effect on health and performance, mediated through
bacterial antagonism, immunomodulation or the histo-
logical alteration of intestinal epithelium. The addition of
probiotics to the diet has been found to improve growth
performance and feed conversion in broilers (Mountzouris
et al., 2007). The mode of action of probiotics in poul-
try includes (i) maintaining normal intestinal microora
by competitive exclusion and antagonism; (ii) altering
metabolism by increasing digestive enzyme activity and
decreasing bacterial enzyme activity and ammonia pro-
duction; (iii) improving feed intake and digestion; and (iv)
neutralizing enterotoxins and stimulating the immune sys-
tem (Jin et al., 1998).
Several substances have been tested as potential alter-
native coccidiostatic feed additives, which might provide
protection against or modulate the effects of coccidial
infections. Information in current literature on the usage of
probiotics as alternatives to coccidiostatic drugs is few and
inconclusive in chicken (Dalloul et al., 2003; Lee et al., 2007)
or in rabbits (Simonova et al., 2009). However, there is no
information in the literature whether probiotics provide
protection against lesions due to Eimeria tenella infection
in chickens.
As a consequence, there is a challenging idea to evaluate
the potential of probiotics in restricting the depression of
performance parameters attributed to coccidiosis. The aim
of our study was to investigate the potential protective use
of 7 probiotic preparations in diets of broiler chickens chal-
lenged with E. tenella, a highly pathogenic Eimeria species
that causes caecal coccidiosis.
2. Materials and methods
This trial was carried out in accordance with the prin-
ciples of regulations of the local Public Veterinary Service
and the Authorities of the Veterinary Faculty of the Univer-
sity of Thessaly. The health of the animals was monitored
by a veterinary surgeon. Moreover birds were vaccinated
against Newcastle disease, Infectious bronchitis and Gum-
boro disease at 12 days of age.
2.1. Experimental design and dietary treatments
A total of 300 day-old Cobb-500 hatched chicks were
randomly allocated into 10 equal groups with 3 replicates
of 10 birds each of equal numbers of males and females.
All replicates were housed in separate oor pens, each
equipped with an infrared lamp with stocking density of
10 birds/m2 . To avoid cross infection, uninfected birds were
kept on the same building but separated room and a xed
protocol of visiting and bird weighing was established by
starting always with non infected birds. Temperature was
gradually decreased from 36 C on day 1 to 24 C on day 21
and then kept constant. The lighting regimen provided 24 h
of continuous light per day until day 2 of experiment and
23 h thereafter.
To meet the nutrient requirements of the broilers
throughout the experimental period, a complete basal diet
was formulated for each of the two stages of growth:
starter and growernisher. The feeds were based on
maizesoybean meal and were formulated to meet starter
(114 days) and growernisher (1542 days) growth
requirements and contained no antibacterial or anticoc-
cidial supplements. Table 1 presents the ingredients and
the composition of the basal diets that were in mash form.
Proximate analysis of three batches of each phase of the
basal diet showed no major deviation from calculated
values. Based on this basal diet, additional experimental
diets were prepared by incorporating either a probiotic
preparation provided by BIOMIN GmbH, Austria, at a
chosen level or lasalocid at 75 mg/kg feed, respectively.
There were ten experimental treatments (groups). Two
of the groups, one challenged with E. tenella and the
other not, were given the basal diet that did not con-
tain any coccidiostatic or other antimicrobial feed additive
and served as controls; untreated uninfected group (UU)
and untreated infected (UI). The third group was chal-
lenged with E. tenella and given the anticoccidial lasalocid
(LasI) at the level of 60 mg/kg feed. The remaining groups
were all challenged with E. tenella, and administered the
basal diet supplemented with different probiotic prepara-
tions: Enterococcus faecium (589) at a low (5 108 cfu/kg
feed; EntLI) or high inclusion rate (5 109 cfu/kg feed;
EntHI); Bidobacterium animalis (503) at 5 108 cfu/kg
feed (BifI); Lactobacillus reuteri 514 at 5 108 cfu/kg feed
(LacI); Bacillus subtilis 588 at 5 108 cfu/kg feed (BacI); the
multi-species probiotic mix PoultryStar (BIOMIN GmbH,
Austria) at low (5 108 cfu/kg diet; PSLI) or high inclusion
rate (5 109 cfu/kg feed; PSHI). Each experimental group
was given the corresponding diet from day 1 to day 42 of
age. Fresh feed was topped up every second day, and ad
libitum availability was ensured throughout for feed and
drinking water.
2.2. E. tenella challenge
Challenge of chickens with E. tenella was carried out
at 14 days of age. To induce sporulation, oocysts were
preserved in 2% potassium dichromate solution, and kept
refrigerated at 35 C until use. Challenge of each bird was
carried out by administering a 2-ml suspension of 2 104
sporulated oocysts of E. tenella directly into the crop via
an oral gavage by a plastic tube. The number of coccidian
oocycsts per milliliter was checked before being used for
oral inoculation of the birds through standard enumeration
techniques.
 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140 33

Table 1
Composition of basal diet (g/kg) in mash form.

Ingredients Starter 114 days of age Growernisher 15 days-slaughter

Maize grains 591.1 g/kg 624.9 g/kg

Soybean meal-46.5 326.2 g/kg 277.3 g/kg
Fish meal 25.0 g/kg 25.0 g/kg
Soy oil 23.0 g/kg 39.0 g/kg
Limestone 18.7 g/kg 17.9 g/kg
Monocalcium phosphate 8.0 g/kg 8.0 g/kg
l-lysine HCl 2.0 g/kg 2.0 g/kg
dl-Methionine 2.0 g/kg 2.0 g/kg
Vitamin premixa 1.0 g/kg 1.0 g/kg
Trace mineral premixb 1.0 g/kg 1.0 g/kg
Salt 2.0 g/kg 1.9 g/kg

Chemical analysisc
Dry matter 882.2 g/kg 884.1 g/kg
Crude protein (N 6.25) 220.2 g/kg 200.2 g/kg
Ether extract 53.3 g/kg 69.5 g/kg
Crude ber 33.0 g/kg 31.1 g/kg
Ash 50.9 g/kg 48.1 g/kg
Starch 366.1 g/kg 370.3 g/kg

Calculated values
Ca 9.3 g/kg 9.0 g/kg
P (total) 6.9 g/kg 6.6 g/kg
Sodium 1.8 g/kg 1.7 g/kg
Chloride 2.4 g/kg 2.3 g/kg
Lysine total 14.0 g/kg 12.7 g/kg
Methionine + cystine 9.8 g/kg 9.3 g/kg
AMEd (kcal/kg) 3100 3200
Crude protein (N 6.25) 220 g/kg 205 g/kg
Ether extract 55 g/kg 75 g/kg
Crude ber 34 g/kg 35 g/kg
a
Supplied per kg of diet: vitamin A, 12,000 IU; vitamin D3 , 5000 IU; vitamin E (-tocopheryl acetate), 30 IU; vitamin K3 , 3 mg; vitamin B1 , 1 mg; vitamin
B2 , 8 mg; vitamin B6 , 3 mg; vitamin B12 , 0.02 mg; nicotinic acid, 20 mg; Ca pantothenate, 20 mg; folic acid, 2 mg; biotin, 0.2 mg; vitamin C, 10 mg; choline
chloride, 480 mg.
b
Supplied per kg of diet: Zn, 125 mg; Mn, 100 mg; Fe, 62 mg; Cu, 7.5 mg; Co, 0.2 mg; I, 2 mg; Se, 0.2 mg.
c
According to AOAC International (1995).
d
Apparent metabolizable energy.

2.3. Performance parameters Oocyst counts were determined in excreta samples

All chicks were individually weighed at the time of allo-
cation into the pens and weekly thereafter. Four hours
prior to bird weighing, diets were removed and feed con-
sumption within each replicate was determined. Feed
conversion ratio values were calculated weekly as the
ratio of feed intake to average weight gain. Mortality was
recorded daily in each replicate.
Seven days after the challenge, (on day 21 of age) the
lesion score was estimated in all groups by evaluating cae-
cal intestinal lesions of 6 chickens per group, by selecting
randomly two birds per replicate. Gross lesion score was
assigned from 0 to 4 according to Johnson and Reid (1970),
where 0 score is the normal status with no gross lesions, 1
score corresponds to small scattered petechiae, 2 to numer-
ous petechiae, 3 to extensive hemorrhage, and 4 score to
extensive hemorrhage giving the caecal intestine a dark
color. Dead birds were given the score of 4.
Existence of bloody diarrhea was determined daily from
day 17 to day 21 of age. The extent of bloody diarrhea was
determined according to Youn and Noh (2001) by assigning
it one of ve levels, where zero is the normal status, and less
than 25%, 2650%, 5175%, or over 75% bloody feces in total
feces, are the remaining levels.
taken daily from each replicate from day 20 to day 26 of age.
Oocyst counts were also determined in the excreta samples
taken from each replicate weekly, at 7, 14, 35 and 42 days
of age. Collection of excreta for oocyst analysis was done
three times daily. Samples from each replicate were placed
in separate airtight plastic bags, homogenized thoroughly
by a domestic mixer, and kept refrigerated until assessed
for total oocyst counts. Homogenized samples were rst
diluted ten fold with tap water and further diluted with
saturated NaCl solution at a ratio of 1:10. Oocyst counts
were determined using McMaster chambers and presented
as the number of oocysts per gram of fresh excreta.
2.4. Histology and morphometric analysis of the intestine
During necropsy of the 6 selected birds on day 21
of age, the gastrointestinal tract was removed and ileal
(from Meckels diverticulum to the ileo-caeco-colic junc-
tion) samples of 1 cm long were taken from the central
part and xed in 10% buffered formalin for histopatholog-
ical evaluation and morphometry under light microscopy.
The intestinal samples were routinely formalin xed, dehy-
drated and embedded in parafn wax. Formalin-xed
intestinal tissues were processed, sectioned at 3 m and
34 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140
stained by hematoxylin and eosin. Histological sections
were examined with a Nikon phase contrast microscope
coupled with a Microcomp integrated digital imaging anal-
ysis system (Nikon Eclipse 80i, Nikon Co., Tokyo, Japan).
Images were viewed (4) to study morphometric param-
eters of intestinal architecture. From the best stained
sections, villous height and crypt depths were measured
manually. For that purpose, three favorably orientated sec-
tions cut perpendicularly from villous enterocytes to the
muscularis mucosa were selected from each animal and
measurements were carried as follows. Villous height (VH)
was estimated by measuring the vertical distance from the
villous tip to villouscrypt junction level for 10 villi per
section. Crypt depth (CD) (the vertical distance from the
villouscrypt junction to the lower limit of the crypt) was
estimated for 10 corresponding crypts per section.
2.5. Enumeration of bacteria populations in ileum and
caecum
Intestinal samples were collected and fresh digesta
samples from ileum and caecum were taken for bacte-
rial analyses within an hour from collection on day 42
from 6 birds per treatment. Digesta samples were serially
diluted in 0.85% sterile saline solution for enumeration of
total aerobes, total anaerobes, Clostridium perfringens, Lac-
tobacillus spp., Bidobacterium spp., E. coli, Enterococcus spp.
and Enterobacteriacae spp. by conventional microbiological
techniques, using selective agar media. All microbiologi-
cal analyses were performed in duplicate and the average
values were used for statistical analysis.
In particular, Lactobacillus spp. were incubated anaer-
obically using MRS agar (Fluka 80961) (pH was adjusted
to 5.5 0.2) for 48 h at 37 C. For the conrmation of Lac-
tobacillus spp. Gram staining (gram positive rod-shaped,
non spore-forming bacilli of various lengths, occasionally
in small chains) and the catalase test (negative reaction)
were performed. Also, in cases of doubt conrmation
of Lactobacillus was performed by using API 50 CH kits
(Biomerieux SA, Marcy-lEtoile/France).
Bidobacterium spp. were anaerobically assayed using
Reinforced Clostridial Agar (RCA) (Fluka 27546) supple-
mented with 0.01 g/ml glucose and adjusted to pH 5
according to Rybka and Kailasapathy (1996). RCA medium
and Prussian blue dye (Fluka 27546 03899) were ster-
ilized separately, cooled mixed aseptically. The Prussian
blue dye was adjusted at a nal concentration of 0.03%
according to Moriya et al. (2006). The media is selec-
tive for Bidobacterium which form white colonies, while
Lactobacillus and streptococci form colonies with a blue
halo and white center. For the conrmation, colonies were
identied as members of the genus Bidobacterium by
the following criteria: they were gram positive, pleomor-
phic rods with characteristic bifurcated Bidobacterium cell
morphology; they were unable to grow under aerobic con-
ditions; they were catalase negative; and they showed
fructose-6-phosphate phosphoketolase (EC 4.1.2.22) activ-
ity as described by Scardovi (1986).
Aerobes were enumerated using Plate Count MUG Agar
(Fluka 80961); the inoculated plates were incubated aero-
bically for up to 48 h at 37 C. Anaerobes were enumerated
by in Reinforced Clostridial Medium (Fluka 27546) and
(Tryptone Sulte Agar without Cyclocerine). The inoculated
plates were incubated anaerobically (in jar) for up to 48 h.
The anaerobic environment was generated by the use of
Anaerocult A (Merck 1.13829) and was conrmed by the
use of Anaerotest (Merck 1.15112). C. perfringens loads
were enumerated by Tryptone Sulte Agar with Cyclocer-
ine.
E. coli were enumerated through the use of Plate Count
MUG Agar (Fluka 80961) (the inoculated plates were incu-
bated aerobically for up to 48 h at 37 C) and TBX Agar
(Fluka 92435) (the inoculated plates were incubated aero-
bically for up to 48 h at 44 C). The conrmation procedures
included the gram stain, the fermentation of lactose in Bril-
liant Green Bile Broth with Lactose 2% (Fluka 16025) and
the production of indole from tryptophan by using DEV
Tryptophan Broth (Fluka 31406).
Enterococcus spp. were enumerated using Slanetz &
Bartley Agar (Fluka 45183) (aerobial incubation at 37 C for
48 h) and Bile Esculin Azide Agar (Fluka 06105) (aerobial
incubation at 37 C for 24 h) for the conrmation of suspect
colonies.
Enterobacteriaceae counts were enumerated using
Violate Red Bile Glucose Agar (VRBGA, Oxoid 485, Bas-
ingstoke, HamPSHire, England) under aerobial incubation.
Enterobacteriacae spp. were enumerated using VRBCA Bac-
teroides.
Anaerobic incubation was achieved using appropri-
ate catalysts (Anaerocult A, Merck, Darmstad, Germany)
in sealed anaerobic jars (Oxoid, Basingstoke, UK). The
conrmation of anaerobic environment was veried by
Anaerotest . Results were expressed as base-10 logarithm
colony-forming units per gram of ileal or caecal digesta.
2.6. Statistical analysis
Experimental data were subjected to analysis of vari-
ance (ANOVA) in the general linear model using the
statistical package of SPSS version 17.00 for Windows
(SPSS, Inc., Chicago, IL, USA). For data on growth per-
formance individual birds were considered to be nested
within pens and data were analyzed by a nested ANOVA
in repeated measurements. Effects of sex and experimen-
tal room were found not to be signicant and not included
in the model. Feed intake and feed conversion ratio values
were analyzed by ANOVA in repeated measurements, the
pen being the experimental unit. Mortality was analyzed by
2 test. As bacterial and oocyst numbers were not normally
distributed, they were log transformed to create a normal
distribution prior to analysis. Levenes test was performed
to check homogeneity of variances and Tukeys test was
carried out to assess any signicant differences at a proba-
bility level of 0.05 among the experimental treatments.
3. Results
3.1. Performance parameters
Table 2 presents the performance data. During the rst
experimental period (014 days), there were no signicant
differences on the performance of the groups fed on any of
 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140 35

the diets. During the challenge phase from 15 to 28 days of

P value

0.271
0.238
0.575
0.474
0.411

0.255
0.101

0.032

0.029

0.025
0.001
0.000

0.000
age, UI group birds ate 1564 g of feed, LasI group 1440 g
and UU group birds 1600 g, respectively. However, feed
intake at 28 days, was not different among the experimen-

0.013

0.018

0.019
0.006

0.008
tal groups (P > 0.05). At the age of 21 days, 7 days after the
0.13
0.15
SEMc

11.4

12.6
19.4
55.4

63.8

64.5
challenge with E. tenella, mean body weight gain of the UI
group turned out to be lower (P < 0.05) than in all other
groups. However at 28 days of age, control UI group pre-
Group 10 PSHI

1.721b
1.501b

1.803b
sented statistically lower body weight compared to UU
0.911

1.293
1.067

1834abc
1175cd
42.6
139.5

birds (P < 0.05) but similar to the other infected groups.

373

659

2565a
Feed intake at 35 days, was higher (P < 0.05) for the PSLI
group compared to groups BifI, LasI and EntLI supple-
mented groups. At the age of 35 days, all probiotic groups
Group 9 PSLI

except EntLI and BifI had higher (P < 0.001) body weight
compared to control UI group. At the age of 42 days, con-
1300ab

1.722b
1.501b

1.804b
1945a

2546a
136.5

1.295
0.912

1.066

trol UU group, and LasI, PSLI and PSHI gave higher (P < 0.05)
43.1

360

707

body weight and body weight gain compared to the UI

group. EntLI, EntHI, BifI, LacI and BacI groups had body
Group 8 BacI

weight values that did not differ among the experimental

groups. The UI had the poorest (P < 0.05) feed conversion
1242abc

2450ab
1.459b

1.718b

1.802b
1904a
133.2

1.294
0.914

1.063

ratio value, compared to all other groups that did not dif-
42.3

343

666

fer among each other. Feed intake was similar among the
groups.
Group 7 LacI

3.2. Eimeria challenge output

1202bcd

1835abc

2403ab
1.719b
1.502b

1.806b
135.5

1.296
0.912

1.066
42.5

659
340

Bloody diarrhea was observed on day 18 of age among

all challenged groups, except for the LasI group where it
Group 6 BifI

appeared one day later (Table 3). On days 19 and 20 of age,

1717bcd

the extent of bloody diarrhea was highest (P < 0.05) in the

2323ab
1.722b
1.503b

1.807b
1100d
137.8

1.292
0.911

1.064

LacI group. BacI and EntLI groups and UI group presented

43.7

634
340

extent of bloody diarrhea that was higher (P < 0.05) than

the BifI and EntHI groups. The LasI, PSLI and PSHI had a
Group 5 EntHI

lower (P < 0.05) extent of bloody diarrhea that did not dif-
(a, b, c, d) Values in the same row with a letter in common do not differ signicantly (P > 0.05).

fer among each other. The UU control group had zero blood
1192bcd

1.699cb
1843ab

2420ab
1.501b

1.805b

in feces. At 28 days of age, 2 weeks after the challenge, mor-

132.4

1.294
1.065
0.910
43.1

353

667

tality in the UU control group was zero. LasI group had one
dead bird. EntLI and EntHI groups, BifI and BacI groups had
3 dead birds, whereas the LacI, PSLI and PSHI had 2 dead
Group 4 EntLI
Cumulative growth performance results on 6 weeks trial with probiotics.a,b

birds. UI control group had the highest mortality with 5

dead birds. PSLI and LacI groups had also low mortality
2346ab
1695cd
1170cd

1.721b

1.810b
1.500b
139.5

1.295
0.912

1.066

compared to BifI and BacI groups (Table 3). The numbers

42.8

354

696

of oocysts per gram of excreta were lower (P < 0.05) in the

LasI group and the LacI, BacI, PSLI and EntLI, EntHI groups.
Results are given as means of groups (n = 3 = subgroups).
Group 3 LasI

BifI group did not have a strong impact on oocyst shedding

1253abc

1830abc

(Table 4) during the challenge phase.

1.652cd
1.456b

1.762b
2546a
133.7

1.265
44.04

0.913

1.064

Groups of chickens fed the corresponding diet.

345

708

3.3. Histopathological lesions and morphometric

analysis of the gut
Group 2 UI

1186bcd
1.641a

1.851a

2.201a

Animals of all groups had a normal ileal structure.

1678d

2171b
128.6

1.343
0.912

1.065
43.3

336

666

The ileal villi were slim and nger-shaped with columnar

standard error of the mean.

epithelium and no histopathological changes were obvious

on the intestinal mucosa (data not shown). Villous height
Group 1 UU

was highest (P < 0.05) in BacI and LacI groups. All other
1.646d
1.459b

1.785b
1361a

1861a

2573a

groups had similar villous height, except for EntLI group

127.6

1.268
0.913

1.061
43.1

347

701

that presented the lowest villous height value among all

the experimental groups (Table 5).
The gross examination of birds showed varying degrees
BW 21

BW 35

BW 42
BW14

BW28
Table 2

BW 7
BW 0

FCR

FCR

FCR

FCR

FCR

FCR

of lesions in the caecum. In cases of mild coccidiosis, birds

a

revealed few scattered to more numerous petechiae on

 36
Table 3
Cumulative Eimeria tenella challenge related results on 6 weeks trial with probiotics.a,b

Group1 UU Group 2 UI Group 3 LasI Group 4 EntLI Group 5 EntHI Group 6 BifI Group 7 LacI Group 8 BacI Group 9 PSLI Group 10 PSHI SEMc P value

Cecal lesion score at 21 days 0.00d 3.33a 1.67c 2.75abc 2.08abc 2.58abc 3.33a 3.08ab 1.75bc 2.17abc 0.23 0.012
Bloody excreta, total 0.00c 3.25a 2.25b 3.25a 3.00ab 3.00ab 4.00a 3.25a 2.25b 2.25b 0.21 0.011
Mortality at 21 days (%)** 0.00c 10.0a 0.00c 3.00b 6.00b 6.00b 3.00b 6.00b 3.00b 3.00b 0.024
Mortality total (%)** 0.00c 20.0a 7.00b 10.0b 13.0b 13.0b 10.0b 13.0b 10.0b 7.00b 0.017

(a, b, c) Values in the same row with a letter in common do not differ signicantly (P > 0.05).
a
Groups of chickens fed the corresponding diet.

I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140

b
Results are given as means of groups (n = 3 = subgroups).
c
Standard error of the mean.
**
Values in the column differ signicantly (P < 0.05) by non parametric analysis of KruskalWallis.

Table 4
Oocyst output after Eimeria tenella challenge transformed in a Log10 base.a,b

Log10 Oocyst counts/g excreta SEMc P value

Group 1 UU Group 2 UI Group 3 LasI Group 4 EntLI Group 5 EntHI Group 6 BifI Group 7 LacI Group 8 BacI Group 9 PSLI Group 10 PSHI

Day 7 of age 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Day 14 of age 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
D5 post inf = day 19 of age 0.00c 3.17a 0.00c 2.71ab 2.66ab 2.89ab 2.54ab 2.36b 2.43ab 2.29b 0.11 0.000
D6 pi 0.00d 4.05a 2.73c 3.55ab 3.47ab 3.49ab 3.43b 3.38b 3.47ab 3.54ab 0.21 0.000
D7 pi 0.00g 5.46a 3.55c 5.17b 5.05bc 5.23ab 4.74d 4.25e 4.81cd 5.00bcd 0.12 0.000
D8 pi 0.00g 5.17a 3.23c 4.97abc 4.50d 5.07ab 4.09e 4.15e 4.67cd 4.77bcd 0.13 0.000
D9 pi 0.00g 5.01a 3.03c 4.93ab 4.03de 4.34cd 3.85e 3.93e 4.59bc 4.77ab 0.14 0.000
D10 pi 0.00c 4.36a 3.04e 3.55cd 3.77cd 3.85c 3.45d 3.88c 3.94bc 4.33ab 0.11 0.000
D11 pi 0.00c 4.18a 2.76e 3.36cd 3.78ab 3.61bc 3.03de 3.85ab 3.85ab 4.13a 0.12 0.000
D12 pi 0.00e 3.94a 2.66d 3.24bc 3.75a 3.61ab 3.06cd 3.45abc 3.72ab 3.81a 0.14 0.000
Day 28 of age 0.00d 3.65a 2.73c 3.04bc 3.48ab 3.48ab 2.81c 2.75c 3.46ab 3.45ab 0.11 0.000
Day 35 of age 0.00d 3.36a 1.96c 2.74ab 2.75ab 2.95ab 2.70ab 2.66b 3.21ab 2.62bc 0.12 0.000
Day 42 of age 0.00c 3.23a 0.00c 2.69ab 2.49ab 2.85ab 2.39b 2.54ab 2.36b 2.46b 0.11 0.000

(a, b, c, d, e, f) Values in the same row with a letter in common do not differ signicantly (P > 0.05).
a
Groups of chickens fed the corresponding diet.
b
Results are given as means of groups (n = 3 = subgroups).
c
Standard error of the mean.
 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140 37

the mucosal and serosal surface of caecum. In birds with

P value

0.043
0.095
0.308
severe coccidiosis, ballooned caecum, hemmorhagic spots
on caecal wall, enlargement of caecum with consolida-
tion of caecal contents were observed. The caecal contents

3.62
0.24
SEMc

20.1
showed clotted blood and solid caste of necrotized blood
cells, epithelial cells and other debris, and some times
Group 10 PSHI change in coloration from reddish to milky white. Marked
thickening of the caecal wall was found and the serosa
729.5cd
96.7bc
showed petechiae to diffuse hemorrhages and in some
7.54
cases erosions. These lesions were all absent in the unin-
fected/untreated group.
Group 9 PSLI

Histopathological lesions of caecum in all groups except

110.8abc

UU group, revealed numerous epithelial cells parasitized by

891.4b

8.04

E. tenella oocysts. Also, numerous oocysts and sporocysts

of E. tenella were present in the lumen of caecum. Atro-
Group 8 BacI

phy of villi due to loss of epithelial cells and hyperplasia

of crypts were prominent. In heavy parasitized birds, villi
1021.9a
135.5a

appeared stumpy with squamous epithelium and eroded

7.54

surface. Submucosal edema and congestion of lamina pro-

pria were also observed, and the lamina propria revealed
Group 7 LacI

diffuse lymphocytic inltration.

The caecal lesion scores of the probiotic supplemented
909.3b
123.7a
7.34

groups were not signicantly different from the UI con-

trol group except for the PSLI group. Also, the LasI group
Group 6 BifI

presented signicantly (P < 0.05) lower caecal lesion score

compared to the UI control group but did not differ from
871.0b
127.8a
6.81

EntLI and EntHI groups, BifI, PSLI and PSHI groups. LacI and
BifI groups gave a high lesion score similar to the UI control
Effect of dietary probiotic supplementation on ileum intestinal morphology of broiler chicken at 42 days of age.a,b

Group 5 EntHI

group (Table 5).

117.6ab
831.3bc

3.4. Enumeration of intestinal microora composition

7.06

The composition of the ileal microbiota of chickens at

Group 4 EntLI

the end of the experiment is shown in Table 6. In the

ileum, the Lactobacillus counts were higher (P < 0.05) in the
677.4d
131.2a

LacI group and lower (P < 0.05) in BifI and PSHI groups.
(a, b, c, d) Values in the same row with a different letters differ signicantly at (P > 0.05).
5.16

Total aerobes were higher (P < 0.05) in the LasI group and
lower (P < 0.05) in EntHI group. Total anaerobes counts
Group 3 LasI

were higher (P < 0.05) in the LacI group and lower (P < 0.05)
820.3bc

in PSHI group. Bidobacterium spp. were higher (P < 0.05)

90.5c

in the LasI group and lower (P < 0.05) in EntHI group. Enter-
9.05

obacter spp. were found to be higher (P < 0.05) in the LasI

group and lower (P < 0.05) in EntHI and PSHI groups. The
Group 2 UI

772.7bc

ratio of E. coli populations to Lactobacillus spp. was lower

Results are given as means of groups (n = 3 = subgroups).
92.3bc
8.37

(P < 0.05) in groups PSLI, PSHI, LacI and UU groups com-

pared to control infected UI groups (0.29, 0.30, 0.36 and
Groups of chickens fed the corresponding diet.
Group 1 UU

034 vs. 0.51; respectively). In the caecum, the Lactobacil-

lus counts were numerically higher in the LacI group and
859.7b
124.1a

lower (P < 0.05) in LasI group. Enterococcus counts were

6.92

higher (P < 0.05) in the LacI and UU groups and lower

(P < 0.05) in PSHI group. Enterobacter spp. was found to be
Villous height to crypt depth ratio

higher (P < 0.05) in the LasI group and lower (P < 0.05) in
Standard error of the mean.

PSLI group. The ratio of E. coli populations to Lactobacillus

spp. was lower (P < 0.05) in group PSLI (0.26) and higher
(P < 0.05) in group LasI (0.64), while PSHI, EntLI and BacI
Villous height (m)
Crypt depth (m)

groups presented relatively low ratio value (0.39).

4. Discussion
Table 5

Ileum

Coccidiosis is mainly controlled by the use of

a

chemotherapeutic agents; however novel approaches are

 38 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140

P Value urgently needed due to the increasing emergence of drug-

0.033
0.012
0.038
0.043

0.017

0.047
0.019
0.029
0.025
0.016
0.045
0.028
0.044
0.008

0.003
0.000
resistant parasite strains in commercial poultry production
settings (Chapman, 1997; Williams, 2002). Feeding natural
dietary supplements or probiotics to animals to enhance
Pooled SEMc

their innate defence mechanisms could effectively reduce

or even prevent the need for therapy of these enteric infec-
0.155
0.138

0.117
0.188
0.363
0.171
0.134
0.065
0.035
0.041
0.063
0.088
0.037

0.052
0.022

0.078
tions. Although a variety of natural products have been
investigated in search for alternative controls of coccidio-
Group 10 PSHI

sis in chickens (Dalloul and Lillehoj, 2006; Wallace et al.,

2010), the effects of probiotics on E. tenella infection has

8.58abcd

3.43bcd
not been studied, in details. The present work investigated

3.93de
5.88ab

3.18ab

7.89ab

3.43b
4.15e

3.73e

8.43a
6.42a
6.60a
6.78c

2.01c
7.22

7.48
the effects of various probiotic-based diets on E. tenella
infection.
Group 9 PSLI

Prophylactic medication has been used to control

8.44abcd

and prevent coccidiosis in commercially grown chickens.

3.96cde
7.27ab
7.59ab

2.87ab

7.88ab

5.42ab

3.64ab
5.66bc

2.15bc

7.00bc
4.83d

Assessment of host disease susceptibility to avian coc-

2.15e
3.80e
7.17a

8.16a

cidiosis has been evaluated by enumerating fecal oocysts

and body weight changes following challenge infection
Group 8 BacI

with live coccidia parasites (Chapman et al., 2005). The

8.37bcde
4.18bcd

4.25cde
7.27abc

5.51abc

7.16abc

anticoccidial effect of probiotics was assessed using fecal

Effect of dietary probiotic supplementation on ileum and caecum bacteria populations (log cfu/g digesta) of broiler chicken at 42 days of age.a,b

7.34ab

5.88ab

8.23ab

5.53ab
3.07ab

3.33cd
2.72b

3.52b
8.41a
7.08a

oocyst shedding and body weight gains. Supplementa-

tion of chickens with probiotics reduced modestly fecal
oocyst output in chickens infected with E. tenella, although
Group 7 LacI

the same treatment signicantly affected body weight

4.46ab

8.41ab
8.77ab
7.73ab

4.75ab
7.40ab

3.06ab

5.60ab
5.49bc

loss caused by coccidiosis. The underlying immunological

2.72b

3.50b
7.76a

7.36a

8.85a

4.42a
5.90a

mechanism responsible for probiotic-mediated protection

against coccidiosis is not known. However, many cytokines
Group 6 BifI

are known to mediate protective cell-mediated immune

4.25bcd
5.78abc

response against intracellular pathogens including coccid-

7.49ab

5.58ab

8.52ab

5.84ab
4.14ab
4.73ab
2.30bc
6.98b

6.68a

3.73a

9.13a
7.95a
8.34a

4.23a

ian (Choi et al., 1999).

In this study, the inuence of various probiotic-
supplemented feeds given to broiler chickens, experimen-
Group 5 EntHI

tally infected with E. tenella, was evaluated and compared

4.48bcd
4.30bcd
8.54abc
7.50abc

to non infected and infected birds fed with the convention-

8.17ab

7.95ab
5.59ab
2.80ab
5.03cd
6.92bc
6.86b

2.74b

3.29b
3.69e
6.97a
5.39c

ally recognized anticoccidial drug lasalocid. Major target in

this study was to nd a natural product with anticoccidial
(a, b, c, d) Values in the same row with a different letter differ signicantly at (P > 0.05).

properties that could be used as a feed additive without

Group 4 EntLI

withdrawal period or any adverse effects that may be asso-

3.99bcd

4.29bcd
8.67abc

ciated with anticoccidial drugs.

7.36ab

5.84ab

7.83ab

5.60ab
2.48bc
7.03bc

4.75d

3.16d
2.39b

7.83b

3.15b
6.75a

8.02a

Adverse effects of coccidiosis due to E. tenella, include

bloody diarrhea, intestinal lesions, depressed growth rate
Group 3 LasI

and, sometimes, high mortality (Giannenas et al., 2004).

Therefore, a number of performance parameters includ-
5.17bcd

8.00cde
7.47ab

3.45ab

8.29ab

4.23ab
7.07bc
6.58b
4.85b

3.36b

ing body weight gain, feed intake, feed conversion ratio,

7.55a

6.12a
7.22a

4.84a

4.83a
4.02a

Results are given as means of groups (n = 3 = subgroups).

mortality, along with caecal lesion score, bloody diarrhea

and oocysts output of the experimental groups were inves-
Group 2 UI

Groups of chickens fed the corresponding diet.

tigated after challenging chickens at 14 days of age with

4.24cde
5.81abc

4.07abc
7.89de
2.74ab

7.65ab

5.53ab
7.20ab
6.94bc

4.32bc

sporulated oocysts of E. tenella. We also evaluated intesti-

4.90d

3.44b
7.24a

3.69a

8.55a

8.08a

nal integrity and ileal and caecal microora composition,

in order to assess any effects of dietary probiotic supple-
Group 1 UU

mentation.
4.70abc

Dalloul et al. (2003) found that Lactobacillus-based pro-

3.90de
7.37ab

5.87ab

8.54ab
2.39bc
7.01bc

3.44b
2.50b

7.72e
6.87a
5.93a

8.19a
6.47a
4.39a
6.69c

Standard error of the mean.

biotics gave positive results on performance of Eimeria

acervulina challenged chickens. These authors demon-
Clostridium Perfrigens

Clostridium Perfrigens

strated an immunoregulatory effect of a dietary probiotic

Bidobacterium spp.

Bidobacterium spp.

on the local immune system in broiler chickens (e.g.,

Enterobacteriacae

Enterobacteriacae
Lactobacillus spp.

Lactobacillus spp.
Total anaerobes

Total anaerobes

IEL), reduced oocyst shedding, and a rationale for fur-

Total aerobes

Total aerobes
Enterococci

Enterococci

ther study of their benecial effects in order to elucidate

Caecum

their protective role. In a similar study, Lee et al. (2007)

E. coli

E. coli
Table 6

Ileum

concluded that a Pediococcus acidilactici probiotic prod-

a

uct (MitoGrow) enhanced the resistance of birds and

 I. Giannenas et al. / Veterinary Parasitology 188 (2012) 3140
partially protected them against coccidiosis. However, the
mechanistic details mediating such protection are not
fully understood and remain to be claried, especially
in the light of the wide array of immune cells acti-
vated by probiotic bacteria. In particular, these authors
suggested that an analysis of the different cytokines
and chemokines induced by feeding Pediococcus that
would provide valuable new information on its protec-
tive immunity to coccidiosis due to different species
of Eimeria, such as E. tenella, E. acervulina and E. max-
ima.
Gut microora has signicant effects on host nutri-
tion, health, and, growth performance (Barrow, 1992) by
interacting with utilization and the development of the
gut system of the host. This interaction is very complex
and, depending on the composition and activity of the gut
microora, it can have either positive or negative effects
on the health and growth of birds. For example, when
pathogens attach to the mucosa, gut integrity and func-
tion are severely affected (Droleskey et al., 1994) and
the immune system is challenged (Neish, 2002). Chickens
kept in a pathogen-free environment grow 15% faster than
those grown under conventional conditions where they
are exposed to bacteria and viruses (Klasing, 1987). Fur-
thermore, it is generally accepted that gut microora is a
nutritional burden in fast-growing broiler chickens, since
an active microora component may have an increased
energy requirement for maintenance and a reduced ef-
ciency of nutrient utilization.
The structure of the intestinal mucosa can reveal
some information on gut health. In our study, mucosal
architecture and crypt depth was inuenced by different
diets. Stressors that are present in the digesta can lead
relatively quickly to changes in the intestinal mucosa,
due to the close proximity of the mucosal surface and the
intestinal content. Changes in intestinal morphology, such
as shorter villi and deeper crypts have been associated
with the presence of toxins (Yason et al., 1987) or higher
tissue turnover (Miles et al., 2006). In the present study,
an increase in ileal villus height and crypt depth ratio
in the ileum of the Lactobacillus fed chickens was found
compared to control birds. Demand for energy and protein
for gut maintenance is high compared with other organs.
Cook and Bird (1973) reported shorter villi and deeper
crypts when the counts of pathogenic bacteria increase
in the gastrointestinal tract, which resulted in fewer
absorptive and more secretory cells.
The results of our study indicated that probiotic
strain-supplemented broilers, even in the event of E.
tenella-infection, show better performance parameters
than non supplemented ones, approaching those of con-
ventionally treated or non-infected broilers. Furthermore,
there is a differentiation amongst the various probi-
otic strains, regarding their benecial/inversive effect on
coccidia-derived performance impairment. Lactobacillus
supplementation had a high impact on oocyst shedding.
The groups given the combination of probiotics at both
low and high supplementation level had a good impact
on caecal lesion score. PSHI group had lowest mortal-
ity. Lactobacillus and Bacillous probiotic supplementation
gave higher villous height. Lactobacillus also improved
39
Lactobacillus and Bidobacterium populations in both the
ileum and caecum.
The idea of using probiotics to modulate avian coccidio-
sis is a novelty in animal nutrition and may become easily
popular, because they are not synthetic products. However,
they have to be extensively investigated in terms of their
mechanisms of action and efcacious level of administra-
tion alone or in combination with other probiotics such as
multi-strain products with effective antiprotozoal effects
or in combination with other natural feed additives with
ascertained antiparasitic activity such as plant based or
organic acid containing products.
In conclusion, the results of the present study suggest
that in the absence of in-feed anticoccidial drugs, treat-
ment with probiotics could alleviate the impact of parasite
infection on chicken by exerting a coccidiostatic effect
against E. tenella may help to maintain enteric health and
minimize the risk and spread of coccidiosis. Effect of probi-
otics in oocyst shedding was signicantly lower than that
exhibited by lasalocid. However, bearing in mind that in
many parameters probiotic multi-strain supplementation
was not signicantly worse than lasalocid in terms of BW
growth, feed to gain ratio and intestinal lesion scoring, we
would suggest that further research is needed to examine
the effective dosage, to identify the active components and
to elucidate their mechanism of action.
Acknowledgment
Authors are grateful to Ilias Kyriazakis for commenting
on earlier versions of the manuscript.
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