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The e ec s o a compe i i e exclusion p oduc and
wo p obio ics on Salmonella coloniza ion and
nu ien diges ibili y in b oile chickens
C. Schnei z,∗,1E. Koi unen,†P. Tuunainen,†and J. Valaja†‡
∗O ion Co po a ion, P.O.Box 425, 20101 Tu ku, Finland; †MTT Ag i ood Resea ch
Finland, Animal P oduc ion Resea ch, 31600 Jokioinen, Finland; and ‡Depa men o
Ag icul u al Sciences, P.O. Box 28, 00014 Uni e si y o Helsinki, Finland
P ima y Audience: quali y assu ance pe sonnel, e e ina ians
SUMMARY
Compe i i e exclusion (CE) cul u es, gi en as a single dose on he day o ha ch, oge he wi h
good hygienic p ac ices has been shown o be a no el app oach o con ol Salmonella in poul y.
The abili y o he CE p oduc B oilac and 2 p obio ics, Flo aMax-B11 and Colos um, o
p e en Salmonella coloniza ion in newly ha ched chickens was e alua ed employing a sligh ly
modi ied Mead-model chicken assay. In a pa allel s udy he e ec o he 3 ea men s on he
p oduc ion o ola ile a y acids in he ceca we e de e mined. In he Salmonella s udy 2 sepa a e
expe imen s we e done. In he i s expe imen all 3 ea men ma e ials we e gi en as a single
dose on d 1. In he second expe imen , which consis ed only o B oilac and Flo aMax-B11,
he la e was gi en in he d inking wa e du ing he 3 i s d a e ha ch. In bo h expe imen s
he chicks we e challenged wi h Salmonella en e ica se o a In an is on d 2. The esul s o he
p esen s udy show ha B oilac was supe io o he 2 o he ea men ma e ials in p o ec ing he
newly ha ched chickens agains Salmonella coloniza ion. The pa allel s udy showed only mino
di e ences among he di e en ea men s. Based on he esul s o he Salmonella challenge
s udy, i was concluded ha B oilac was he only ea men ma e ial ha was es ablished in
he gu o he newly ha ched chickens in such a way ha he coloniza ion o Salmonella was
p ohibi ed.
Key wo ds: compe i i e exclusion, b oile , Salmonella, bi d pe o mance
2016 J. Appl. Poul . Res. 25:396–406
h p://dx.doi.o g/10.3382/jap /p w025
DESCRIPTION OF PROBLEM
Compe i i e exclusion (CE) cul u es, gi en as
a single dose on he day-o -ha ch, oge he wi h
good hygienic p ac ices has been shown o be
a no el app oach o con ol Salmonella in poul-
‡Cu en add ess: Depa men o Ag icul u al Sciences,
P.O. Box 28, 00014 Uni e si y o Helsinki, Finland
1Co esponding au ho : [email p o ec ed]
y [1–3]. The ea men has a bene icial e ec
also on hose b oile locks ha ha e been con-
amina ed wi h Salmonella al eady in he ha ch-
e y [4]. Though o iginally de eloped o con ol
Salmonella in ec ions [5], he concep has also
been shown o p o ec chicks agains chicken and
human pa hogenic Esche ichia coli [6–8]andE.
coli ca ying plasmid-bo ne ex ended-spec um
β-lac amases (ESBL) o ans e able class C
se ine β-lac amases (pAmpC enzymes) [9]. In
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SCHNEITZ ET AL.: SALMONELLA COLONIZATION 397
addi ion, he e ec o CE ea men agains
Campylobac e [10–12] and nec o ic en e i is
[13–15] has been shown bo h in small-scale ials
and in he ield. Fu he mo e, i has been shown
ha CE p epa a ions o chicken o igin also p o-
ide p o ec ion agains Salmonella in u key
poul s [16–18], quail [19], and pheasan s [20].
Al eady a an ea ly s age in he his o y
o compe i i e exclusion, an imp o emen in
g ow h a e was obse ed in comme cial b oile
locks ea ed wi h a CE p epa a ion [3,12,
21,22]. Howe e , imp o emen in bi d pe -
o mance is p obably mos appa en in locks
ha a e su e ing om a disease condi ion, e.g.,
om nec o ic en e i is [15]. In a labo a o y-scale
s udy he CE p oduc B oilac was shown o de-
c ease ileal diges a iscosi y, and eed me aboliz-
able ene gy (ME) alue in b oile s ed whea - and
ba ley-based die s [23]. In ano he s udy B oilac
signi ican ly imp o ed o al eed diges ibili y a
35 d when he bi ds we e ed a co n and soybean-
based die . Inc eases in body weigh and ecal
d y-ma e con en we e also obse ed, as well
as an imp o ed eed con e sion a io [24].
P obio ics a e used p ima ily o enhance he
g ow h pe o mance o ood animals o o con-
ol condi ions such as scou ing. They a e gi en
in eed o wa e , o en o e a long pe iod o ime
[25,26]. Imp o emen in weigh gain was shown
when a comme cial p obio ic con aining Lac o-
bacillus acidophilus and L. casei was included
in di e en b oile die s ha we e low in ce ain
nu ien s [27]. In ano he s udy wi h 2 comme -
cial p obio ics consis ing o lac obacilli and ecal
en e ococci, no signi ican di e ences we e ob-
ained in b oile body weigh , eed con e sion o
mo ali y be ween he p obio ic ea men s and
con ol g oup in any o he ials [28]. As wi h
CE p oduc s p obio ics may gi e bes esul when
applied o poul y locks ha a e o some eason
no pe o ming well [29].
Con adic o y esul s ha e been published
also ega ding he an i-Salmonella ac i i y o
p obio ics [17,30–32]. I has been sugges ed
ha he e icacy o p obio ics may depend on
ac o s such as mic obial species composi ion
(e.g., single o mul is ain) and iabili y, admin-
is a ion le el, applica ion me hod, equency o
applica ion, o e all die , bi d age, o e all a m
hygiene, and en i onmen al s ess ac o s [33].
On he o he hand, i has also been sugges ed ha
lac obacilli, which may help o con e p o ec ion
agains Salmonella when added oge he wi h
he o he o ganisms o a poul y cecal suspen-
sion, can make he si ua ion wo se when added
alone [34].
O e he yea s, a ying expe imen al p o-
cedu es ha e been employed o es he e i-
cacy o di e en CE p oduc s and p obio ics
agains Salmonella, which has made he com-
pa ison o hese 2 di icul . This s udy was
unde aken o es he e icacy o he comme -
cial CE p oduc B oilac and 2 comme cial p o-
bio ics, Flo aMax-B11 and Colos um, agains
Salmonella In an is in equal ci cums ances us-
ing a sligh ly modi ied e sion o he chicken
assay sugges ed by Mead e al. [35]. The modi-
ied assay has been desc ibed ea lie by Schnei z
and Hakkinen [36]. Addi ionally, a s udy was
unde aken o e alua e he e ec o he CE
p oduc B oilac and he 2 p obio ics, Flo aMax-
B11 and Colos um, on he nu ien diges ibil-
i y, he AMEno he eed, and he p oduc ion
o ola ile a y acids (VFA), showing es ablish-
men o s ic ly anae obic bac e ia in he chicken
gu when he bi ds a e ed a die based on co n
and soybean meal.
MATERIALS AND METHODS
Tes Ma e ials
B oilac (O ion Co po a ion, Espoo, Finland)
is he i s comme cial CE p oduc . I is a
s ongly selec ed mix u e o bac e ia de i ed
om he cecal con en s o one heal hy adul
g andpa en b eede om 1988. The selec ion
p ocess was based on he abili y o ce ain s ic ly
and acul a i ely anae obic bac e ia o adhe e o
he gu wall o he bi d. The inocula and inal
p oduc ha e been es ed o be ee om poul-
y and human pa hogens and o he unwan ed
bac e ial gene a. B oilac is a eeze-d ied p od-
uc . Flo aMax-B11 (Ve anco SA, Chile 33, Vi-
cen e L´
opez, Buenos Ai es, A gen ina) is also a
eeze-d ied p oduc consis ing o ele en lac ic
acid bac e ial isola es o poul y gas oin es inal
o igin ha belong o 5 di e en Lac obacillus
species. Colos um Liquido (BioCamp Labo a-
o ies L do., Campinas, B azil) is a b o h cul u e
con aining anae obic bac e ia, bac e ia o he
genus En e ococcus and lac ic acid-p oducing
bac e ia om Speci ic Pa hogen F ee (SPF)
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398 JAPR: Resea ch Repo
chicks. Flo aMax-B11 and Colos um Liquido
we e shipped oge he om B azil packed wi h a
coole . The s o age empe a u es o Colos um
Liquido and Flo aMax-B11 ange om +2 o
+8◦C and om +5 o+25◦C, espec i ely. A e
a i al he p oduc s we e s o ed in a e ige a o ,
and hey we e all wi hin expi y when applied o
he chickens. The iabili y o he p oduc s was
no checked because no known disad an ages
had occu ed du ing anspo .
Tes Animals
In he Salmonella challenge s udy, in Expe -
imen I 240 and in Expe imen II 180 newly-
ha ched Ross 508 b oile chickens we e b ough
om a comme cial ha che y and di ided an-
domly in g oups o 10 and ea ed on so wood
g anula ed bedding o aspen in solid-bo om
ca dboa d boxes.
In he diges ibili y s udy, a o al o 192 newly-
ha ched Ross 508 b oile male chicks we e di-
ided in o 4 expe imen al g oups, six eplica es
pe ea men . In he beginning o he s udy he e
we e 8 b oile chickens pe cage and eplica e.
All expe imen al p ocedu es we e app o ed by
he Na ional E hical Commi ee o Animal Ex-
pe imen s (H¨
ameenlinna, Finland).
Feed and Wa e
In he Salmonella challenge s udy, he chicks
we e gi en a comme cial eed, B oile Pikku
Punahel a (Suomen Rehu, Hankkija-Maa alous
Oy, Finland). The eed did no con ain any an-
ibac e ials o an icoccidials. Regula ap wa e
was applied om wa e ing bo les.
In he bi d diges ibili y s udy, an expe imen-
al die wi hou g ow h-p omo ing an ibio ics o
coccidios a s was o mula ed o achie e he nu-
ien equi emen s o Ross-508 b oile chick-
ens (Table 1). G ain ing edien s o he die
we e g ound in a olle mill. Feeds we e mixed
and s eam-pelle ed (KAHL 33–50, AMANDUS
KAHL GmbH & Co. KG, Hambu g, Ge many).
The pelle diame e was 4 mm. Feeds and wa e
we e a ailable ad libi um h oughou he expe i-
men s excep du ing he as ing pe iod. Ti anium
oxide (Ti) was used as an indica o in he die o
de e mine he diges ibili ies, AMEn, and e en-
ion o ni ogen.
Table 1. The composi ion and nu ien con en s o he
expe imen al die in he diges ibili y s udy.
Ing edien s, g/kg
Co n 558.8
Soybean meal 350.6
Rapeseed oil 43.8
Monocalcium phospha e 19.5
Limes one 10.0
NaCl 3.8
Mine al p emix12.0
Vi amin p emix22.0
DL-Me hionine 1.6
L-Lysine 3.1
L-Th eonine 0.9
Ti anium oxide 4.0
Nu ien con en , g/kg DM (excep DM and AMEn)
DM, g/kg 890,9
AME, kcal/kg33487.1
C ude p o ein 235.5
C ude a 79.4
C ude ibe 30.0
Ash 69.6
Lysine415.83
Me hionine45.73
Th eonine410.61
Calcium410.84
Phospho us (a ailable)45.08
1P o ided pe kilog am o he comple e die : Ca 0.63 g, i on
29.1 mg, coppe 8.0 mg, manganese 50.3 mg, zinc 65.1 mg,
iodine 0.51 mg, selenium 0.20 mg.
2P o ided pe kilog am o he comple e die : Ca 331 g, i-
amin A 6.00 IU, i amin D32.25 IU, i amin E 30,000 mg
(α- oko e ol 27 270 mg), i amin K31,505 mg, i amin
B11,257 mg, i amin B23,000 mg, i amin B62,010 mg,
i amin B12 12.5 mg, bio in 75 mg, olic acid 504 mg, niacin
20,072 mg, pan o henic acid 7,506 mg.
3Based on chemical analysis o eed ing edien s.
4Based on alues o eed ing edien s in Feed ables and nu-
ien equi emen s (MTT Ag i ood Resea ch Finland, 2012).
Expe imen al Design
The Salmonella challenge s udy included 2
expe imen s. Expe imen I consis ed o 3 sepa-
a e ials wi h 80 chickens in each ial, 20 bi ds
in 2 equal g oups pe ea men . Tube and sy inge
we e used o deli e by o al ga age he dosages
sugges ed by he manu ac u e s as a single dose
on he day o ha ch in a dose olume o 0.3 mL:
1 mg o B oilac (5 g o 5,000 chicks) in phos-
pha e bu e ed pep one wa e , 6 mg o Flo aMax-
B11 (60 g o 10,000 chicks) in skimmed milk
[37], and 0.01 mL o Colos um in egula ap
wa e (50 mL o 5,000 chicks).
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SCHNEITZ ET AL.: SALMONELLA COLONIZATION 399
Expe imen II consis ed also o 3 ials, bu
included only B oilac and Flo aMax-B11. This
expe imen was conduc ed, because Flo aMax-
B11 was ins uc ed o be gi en in he d inking
wa e du ing a ime pe iod o 3 consecu i e d.
The dosing was, howe e , di icul o adjus , be-
cause i was by way o he d inking wa e . Thus,
he o al doses pe chicken we e 35.9, 28.3, and
30.8 mg in he 3 ials, espec i ely. B oilac was
gi en pe os on he day o ha ch and dosed as
desc ibed in Expe imen 1.
Twen y- ou hou s a e dosage, he chicks
in Expe imen I we e challenged ia o al ga -
age (0.5 mL) wi h 1,300 o 2,300 and in
Expe imen II wi h 1,400-3,600 CFU/chick o
a nalidixic acid esis an de i a e o Salmonella
In an is. The bi ds we e humanely eu hanized 5
d la e wi h ca bon dioxide gas, hei ceca we e
emo ed and Salmonella was cul i a ed om
hei cecal con en s bo h quan i a i ely and by
en ichmen as desc ibed by Schnei z and Hakki-
nen [35].
In he bi d diges ibili y s udy he e we e 4
ea men g oups: 1) un ea ed con ol; 2) 1 mg
o B oilac in a dose olume o 0.3 mL pe bi d;
3) 6 mg o Flo aMax-B11 in a dose olume o
0.3 mL pe bi d and; 4) 0.01 mL o Colos um
in a dose olume o 0.3 mL pe bi d. The p od-
uc s we e gi en as a single dose on he day o
ha ch. Bi ds we e andomly assigned o ea -
men s g oups. T ea men g oups we e placed
sepa a ed in a 3- ie ed ba e y (6 cages pe g oup
wi h 8 chicks in each cage). The cage wi e bo -
oms allowed passage o eces and o o al col-
lec ion o he eces pla es we e placed unde he
cages.
A he ages o 12 and 23 d, 2 chicks pe cage
we e humanly eu hanized by ce ical disloca-
ion. The in es inal con en s om he ileum we e
collec ed o de e mine ileal iscosi y. Ileal di-
ges a samples we e cen i uged (12,000 ×g,
3 minu es) and he iscosi y was measu ed us-
ing a B ook ield DV-II+Cone and Pla e P o-
g ammable Viscome e (B ook ield Enginee -
ing Labo a o ies Inc., Middlebo o, USA). The
cone used was CPE-40.
A he age o 26 o 29 d, eces we e collec ed
om he pla es unde he cages o de e mine
AMEn, o al ac diges ibili y o o ganic ma -
e and ni ogen. The 24-hou eed wi hd awal
pe iod was s a ed a he age o 29 d and a e
ha eed was gi en again ad libi um. Fou hou s
a e s a ing he eeding he 4 emaining chicks,
a he age o 30 d, om each cage we e hu-
manely killed by ce ical disloca ion o collec
ileal and cecal con en s. The ileal and cecal con-
en s, espec i ely, om bi ds in each cage we e
pooled. The ileal con en s we e eeze-d ied and
s o ed in a e ige a o o la e use. The ileal
diges ibili y o p o ein and o ganic ma e we e
measu ed om ileal con en s and he diges a pH
and concen a ions o VFA and lac ic acid we e
measu ed om he esh cecal con en s.
Chemical Analysis
Feed samples o analysis om he eady-
mixed ba ch we e aken o he basal expe imen-
al die , and passed h ough a hamme mill i ed
wi h a 1-mm mesh. D y ma e con en , c ude
a and ash we e de e mined by s anda d me h-
ods [38]. C ude ibe was de e mined wi h he
modi ied me hod AOAC (me hod 962.09) us-
ing glass wool ins ead o a ce amic ibe il-
e . Ni ogen con en was analyzed using he
Leco FP 428 ni ogen analyze (Leco Co po-
a ion, S . Joseph, MI). C ude p o ein con en
was calcula ed by mul iplying he ni ogen con-
en by 6.25 [p o ein is 16% ni ogen (100/16 =
6.25)]. Ene gy alue in Table 1(kcal/kg AME)
is based on he chemical analysis o eed in-
g edien s. The g oss ene gy (GE) o he eed
and la e men ioned eces was measu ed wi h
Pa 6200 Oxygen Bomb Calo ime e (Pa In-
s umen Co. Moline, IL 61265). The pH was
de e mined wi h Me le Toledo 345 pH me e
(Me le -Toledo AG, Schwe zenbach, Swi ze -
land), bu be o e ha , he cecal sample was di-
lu ed 1:3 (weigh /weigh ) wi h deionized wa e .
To exclude c ude ma e ial, he sample was cen-
i uged a 3,000 pm o 10 minu es (He aeus
Mul i uge). The de e mina ion o lac ic acid was
pe o med colo ime ically om he supe na an
[39,40]. Fo de e mina ion o VFA, he supe -
na an was il e ed (ch oma il GF/PET-20/25)
and u he dilu ed 1:5 wi h deionized wa e and
mixed. Two hund ed mic oli e o he dilu ed su-
pe na an , 50 μL o o mic acid (98%) and 50 μL
o sa u a ed me cu ic chlo ide (8 g/100 mL)
we e mixed in a 25-mL measu ing bo le. The
de e mina ion was done using a gas ch oma o-
g aph (model 6890, Hewle -Packa d, Wilming-
on, DE) wi h an au oma ic injec o HP 7683,
FID de ec o , spli injec ion po , and a silica
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400 JAPR: Resea ch Repo
capilla y column (10 m ×0.53 mm). The ca ie
gas was helium ( low 7 ml/min). In he de e mi-
na ion o he esul s, an ex e nal s anda diza ion
was used.
Calcula ions and S a is ical Analysis
The ollowing o mula was used o calcula e
appa en diges ibili y o he die [23]:
Diges ibili y (%)=100 −[100
×(Die a y i anium oxide con en /
Fecal o ileal i anium oxide con en
×Fecal o ileal nu ien con en /
Die a y nu ien con en )]
AME was calcula ed acco ding o Ame ah e
al. [41] using he ollowed o mula:
AME (kcal/kg die DM)=( eed in ake ×GEdie )
−(exc e a ou pu ×GEexc e a)/ eed in ake
whe e, GE =The g oss ene gy o die o exc e a
AMEn alues we e de e mined by co ec ion
o ze o ni ogen e en ion by simple mul iplica-
ion wi h 8.73 kcal/g o ni ogen e ained in he
body [41].
The esul s o bi d diges ibili y s udy we e
calcula ed using he ANOVA in GLM P ocedu e
o SAS (SAS Ins i u e Inc., Ca y, NC, USA).
The pa ame e s p esen ed in Tables 4and 5we e
analyzed using he ollowing model: Yij =μ+
i+εik,whe eYij =obse a ion, μ= he gen-
e al mean, i= he e ec o he ea men (i=
1,. . . .4), and εijk = he expe imen al e o e m.
The Tukey’s ange es was used as a single-s ep
mul iple compa ison p ocedu e and as a s a is-
ical es o compa e di e ences be ween means
(ls-means). P≤0.05 was conside ed o be sig-
ni ican .
RESULTS AND DISCUSSION
Mead e al. [35] desc ibed a chick assay o
s anda dize he me hod used o e alua e he e -
icacy o CE p epa a ions agains Salmonella.
Newly-ha ched chicks a e ea ed o ally on d 1,
challenged o ally 24 h la e wi h Salmonella and
examined i e d pos challenge o de e mine bo h
he p opo ion o posi i e bi ds in ea ed and
con ol g oups and he le els o Salmonella ca -
iage in in ec ed indi iduals. The e icacy o he
ea men is de e mined by calcula ing an In ec-
ion Fac o (IF) alue, which is he geome ic
mean o he numbe o Salmonella o ganisms
pe g am o cecal con en s o all chicks in a pa -
icula g oup (IF =log10CFU g−1). A P o ec ion
Fac o (PF) alue is ob ained by di iding he IF
alue o he con ol g oup by ha o he ea ed
g oup [35,42]. A PF alue o 4.0 has been sug-
ges ed as he lowes limi o accep ance o a CE
p epa a ion o use in he ield. Howe e , a be e
way o e alua e he e icacy o a ea men ma e-
ial may be o use he di e ence be ween he IF
alues o con ol and ea ed g oups (di e ence
=IF alue) [36]. In his s udy we examined he
esul s in he same way, as desc ibed by Schnei z
and Hakkinen [36], because s a is ical e alua-
ion is no needed when he di e ences in he
e icacy be ween he di e en ea men g oups
is mani es .
The esul s o he Salmonella challenge s udy
a e p esen ed in Tables 2and 3. Each IF alue
p esen ed in Tables 2and 3is a mean o 20 chicks
(2 g oups o 10 chicks each). In Expe imen I
(Table 2), 3 chickens in ials 2 and 3, and in
Expe imen II (Table 3) 2 chickens in ials 2 and
3, died du ing he ea ing pe iod as p esen ed in
he columns in ec ed/all.
The chicks ea ed wi h B oilac we e well
p o ec ed agains he challenge o ganism, he
IF alues in Expe imen 1 being 5.3, 5.0 and
5.0 in he 3 ials, espec i ely. In Expe imen II
he IF alues in he 3 ials we e 4.8, 5.3
and 6.1. When Flo aMax-B11 and Colos um
we e gi en as a single dose on d 1 (Expe i-
men I, Table 2), no e ec could be seen by
hese 2 ea men s. In con as , he e we e highe
loads o Salmonella in 2 ials ea ed wi h
Flo aMax-B11 and in one ial wi h Colos um
han in he co esponding Salmonella con ol
g oups.
In Expe imen II when Flo aMax-B11 was
gi en o 3 d in he d inking wa e , he e
was a sligh educ ion o Salmonella in he
ea ed g oups compa ed o he con ols, he
IF alues being 1.2, 1.1 and 0.5, espec i ely.
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SCHNEITZ ET AL.: SALMONELLA COLONIZATION 401
Table 2. Expe imen I: The e icacy o B oilac , Flo aMax-B11 and Colos um agains Salmonella In an is when
he ea men ma e ials we e gi en as a single dose o day-old chicks.
T ea men ma e ials Salmonella
B oilac Flo aMax-B11 Colos um con ol
T ial IF1IF2in /all3IF IF in /all IF IF in /all IF in /all
1 0.3 5.3 1/20 5.4 0.2 20/20 5.4 0.2 20/20 5.6 20/20
2 0.4 5.0 3/20 5.6 −0.2 19/1945.9 −0.5 20/20 5.4 20/20
3 0.2 5.0 2/20 5.8 −0.6 20/20 4.9 0.3 19/1945.2 19/194
1In ec ion Fac o (IF) is he loga i hmic numbe o colony o ming uni s o Salmonella In an is pe g am o cecal con en s (IF
=log10CFU/g am).
2IF is he di e ence be ween he IF o he con ol g oup and ha o he ea ed g oup.
3The column shows he numbe o Salmonella-posi i e bi ds pe all in ha g oup.
4One chick died du ing he ial pe iod.
Table 3. Expe imen II: The e icacy o B oilac and Flo aMax-B11 agains Salmonella In an is when B oilac was
gi en as a single dose o day-old chicks, and Flo aMax-B11 was gi en in he d inking wa e o 3 d consecu i ely.
T ea men ma e ials
B oilac Flo aMax-B11 Salmonella con ol
T ial IF1IF2in /all3IF IF in /all IF in /all
1 0.0 4.8 0/20 3.6 1.2 19/20 4.8 20/20
20.05.30/19
44.2 1.1 18/20 5.3 20/20
3 0.9 6.1 7/20 6.5 0.5 19/1947.0 20/20
1In ec ion Fac o (IF) is he loga i hmic numbe o colony o ming uni s o Salmonella In an is pe g am o cecal con en s (IF
=log10CFU/g am).
2IF is he di e ence be ween he IF o he con ol g oup and ha o he ea ed g oup.
3The column shows he numbe o Salmonella-posi i e bi ds pe all in ha g oup.
4One chick died du ing he ial pe iod.
Howe e , seeing he high doses o Flo aMax-
B11 consumed by he chickens, he esul is
su p isingly poo . One explana ion may be ha
nei he Flo aMax-B11 no Colos um was able
o block he po en ial a achmen si es on he
gu epi helia well enough o p o ide p o ec-
ion. This lack o a achmen is e iden also
om Expe imen II, in which he chicks we e
gi en Flo aMax-B11 in he d inking wa e o
3 d consecu i ely and he esul s imp o ed only
ma ginally. On he o he hand, as sugges ed by
Ba nes and Impey [34], lac obacilli may p o-
ide p o ec ion agains Salmonella oge he wi h
he o he o ganisms o he cecal suspension, bu
can also make he si ua ion wo se when added
alone. In ou expe ience, lac obacilli, when iso-
la ed om hei na i e en i onmen , lose p op-
e ies such as he abili y o a ach o in es inal
epi helial cells [43]. This loss o wild- ype p op-
e ies seems o be he case also wi h complex,
pu e-cul u e p epa a ions ha in he beginning
ha e been equal in e icacy o mixed cecal cul-
u es [44,45]. I has also been shown ha he
bes p o ec ion agains Salmonella is achie ed
by pu e-cul u e p epa a ions con aining be ween
28 and 50 s ains om 10 di e en gene a [46,
47]. The poo esul s wi h FM-B11 and espe-
cially Colos um Liquido a e di icul o explain
and may be a leas o some ex en due o un-
known mis o une du ing anspo . Howe e , in
Expe imen II, FM-B11 showed some e icacy
agains Salmonella when he p oduc was ed
con inuously o 3 d in high doses.
Because o a ying es ing me hods, he e i-
cacy o di e en p oduc s agains Salmonella is
di icul o compa e. The e icacy o Flo aMax-
B11 agains Salmonella hasbeenshownina
chicken assay model in which he bi ds we e
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402 JAPR: Resea ch Repo
Table 4. The e ec s o B oilac , Flo aMax-B11, and Colos um on pH alue and concen a ions o VFA (μmol/g)
and lac ic acid (μmol/g) in he cecal con en s a 30 d o age.
Con ol B oilac Flo aMax-B11 Colos um SEM
pH 6.2 6.4 6.4 6.3 0.07
Ace ic acid 55.0 59.0 53.8 49.5 3.06
P opionic acid 2.2b7.7a2.5b2.0b0.60
Isobu y ic acid 0.3b0.5a0.4a,b 0.2b0.06
Bu y ic acid 10.9a,b 12.9a11.0a,b 8.6b0.71
Iso ale ic acid 0.3b0.6a0.4a,b 0.2b0.07
Vale ic acid 0.8 1.1 0.9 0.8 0.08
To al VFA 69.4a,b 81.8a67.3b59.7b3.78
Lac ic acid 0.2 0.1 0.2 0.2 0.03
a,bMeans in he same ow ollowed by he same le e s do no di e ; P>0.05 (Tukey’s es ).
i s challenged wi h Salmonella and ea ed 1 h
la e wi h he p obio ic and hei cecal con-
en s a e checked o Salmonella 24 and/o 72
h pos challenge [48,49]. When he chicks we e
i s ea ed wi h Flo aMax-B11 and challenged
24 h la e wi h Salmonella and killed 24 h a e
challenge, 2 ou o 4 ials ailed o wo k [37].
Higgins e al. [37] sugges ha he iming o he
challenge is c ucial. On he o he hand, he na-
u e o his kind o p oduc is p ophylac ic a he
han he apeu ic, so in ha sense hey should
wo k, especially in si ua ions whe e he chal-
lenge comes a e ea men , hough he concep
o CE has been shown o wo k also in si ua ions
whe e he bi ds ha e been con amina ed al eady
in he ha che y [4].
Table 4shows he pH and VFA and lac ic
acid concen a ions in cecal con en s om he
bi d diges ibili y s udy (samples aken a 30 d o
age). The cecal pH alues did no di e be ween
he 3 ea men s. B oilac inc eased (P≤0.05)
he concen a ions o cecal p opionic, isobu y ic,
and iso ale ic acids, as well as he concen a ion
o o al VFA, compa ed o he con ol, bu ha
di e ence was no signi ican (Table 4). Com-
pa ed o Flo aMax-B11 and Colos um, B oilac
inc eased he concen a ion o p opionic acid
and o al VFA (P≤0.05). Fu he , compa ed
o Colos um, B oilac inc eased he cecal con-
cen a ions o isobu y ic, bu y ic, and iso ale ic
acids (P≤0.05). The e was no inc ease in he
concen a ion o o al VFA in he cecal con-
en s o chicks ea ed wi h ei he Flo aMax-B11
o Colos um compa ed o he con ol. Because
bac e ial e men a ion mainly occu s in he ce-
cum [50], only he cecal con en s we e aken o
analysis. In he no mal in es inal lo a, VFA a e
p oduced mainly as a esul o he me abolism
o spo ing and non-spo ing anae obic bac e ia,
and he inc ease in he concen a ions o VFA
is conside ed o be a clea indica o o he es-
ablishmen and g ow h o anae obic bac e ia in
he chicken gu [51,52]. Those VFA ha a e
inhibi o y o Salmonella include ace ic, p opi-
onic, and bu y ic acids [24]. Co ie e al. [53]
no iced a conside able inc ease in p opionic acid
and o al VFA concen a ions in he cecal con-
en s o CE ea ed chicks compa ed wi h he
con ols a 3 d o age, 2 d a e ea men , in-
dica ing ha VFA-p oducing bac e ia p esen in
he ea men ma e ial we e apidly es ablished
in he ceca a e ea men on he day-o -ha ch.
In ano he s udy Nisbe e al. [54] showed ha
CE cul u es ha inc eased (P≤0.05) cecal p o-
pionic acid in 3-day-old chicks, dec eased (P≤
0.05) cecal Salmonella coloniza ion in 10-day-
old chicks compa ed wi h he un ea ed con-
ols, and CE cul u es ha ailed o inc ease
(P≤0.05) cecal p opionic acid concen a ions
in 3-day-old chicks, ailed also o p o ec he
chicks agains cecal Salmonella coloniza ion in
10-day-old chicks. Simila co ela ion be ween
inc easing le els o cecal p opionic acid concen-
a ions and dec easing incidence o Salmonella
in he cecal con en s was also epo ed by Ma in
e al. [55].
Table 5shows he AMEnand he appa en
diges ibili ies and e en ion o nu ien s. The
di e en ea men s had only mino e ec s on
he nu ien u iliza ion and diges a iscosi y.
B oilac inc eased (P≤0.05) he o al ac di-
ges ibili y o o ganic ma e compa ed o ha o
Flo aMax-B11. In addi ion, o al ac diges ibil-
i y o ni ogen was inc eased (P≤0.05) in he
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SCHNEITZ ET AL.: SALMONELLA COLONIZATION 403
Table 5. The e ec s o B oilac , Flo aMax-B11, and Colos um on he ni ogen-co ec ed AME (AMEn) (kcal/kg
DM) diges ibili y o he eed (%), he iscosi y o he ileal con en s (cPs) and ecal d y ma e (%).
Con ol B oilac Flo aMax-B11 Colos um SEM
AMEnMJ/kg DM 3,343.8 3,391.6 3,343.8 3,343.8 14.33
To al ac diges ibili y o o ganic ma e , % 74.2a,b 74.9a73.5b74.1a,b 0.31
To al ac diges ibili y o ni ogen, % 61.2a60.4a,b 58.0b61.3a0.80
Ileal diges ibili y o p o ein, % 82.1 83.3 82.3 82.1 0.50
Ileal diges ibili y o o ganic ma e , % 73.8 74.2 73.9 73.5 0.70
1Ileal iscosi y, 2cPs, a 12 d o age 4.1 4.0 4.0 3.1 0.26
Ileal iscosi y, 2cPs, a 23 d o age 4.4 4.2 3.9 3.0 0.40
Fecal d y ma e , % 27.3 23.5 24.8 26.8 1.27
1T ea men 1: N =5, SEM =1,115∗SEM.
2cPs =Cen ipoise.
a,bMeans in he same ow ollowed by he same le e s do no di e P>0.05 (Tukey’s es ).
Colos um ea ed bi ds bu also in he un ea ed
con ols compa ed o Flo aMax-B11. In he p e-
ious s udy by Schnei z e al. [23] B oilac ea -
men dec eased he ileal iscosi y, bu in he cu -
en s udy he e we e no e ec s on iscosi y by
he di e en ea men s. On he o he hand, he
ileal iscosi y in all ea men g oups was much
lowe han in he p e ious s udy. In es inal is-
cosi y is known o be a majo ac o limi ing bi d
pe o mance [56]. Inc easing iscosi y educes
he mixing and eed passage a e [57]. The com-
posi ion o he eed is known o a ec he iscos-
i y in he small in es ine. Soluble a abinoxylans
in ye and whea and β-glucans in ba ley ha e
shown o gi e ise o highly iscous condi ions in
he small in es ine o chicks [56,58,59].Acco d-
ing o Rod iguez e al. [60] diges a iscosi y a
he jejunal le el was signi ican ly highe in bi ds
ecei ing he die based on whea and ba ley han
in bi ds ed co n based die .
The in es inal mic o lo a o he chick changes
wi h age, many o he s ic ly anae obic s ains
appea ing only a e wk 2 and wk 3 o li e
[61–63]. The e a ded de elopmen o he in-
es inal mic o lo a makes he young chicken ul-
ne able o en e opa hogens such as Salmonella
[5]. I has been shown, ha newly-ha ched chick-
ens a e ela i ely well p o ec ed agains an o al
Salmonella challenge, al eady a couple o hou s
a e ea men wi h a CE cul u e [64,65]. Be-
cause he e ec is so apid, p o ec ion is hough
o be p ima ily physical [66]. The na i e mi-
c o lo a blocks he po en ial a achmen si es on
he gu epi helia, hus inc easing esis ance o
Salmonella. The esul s o he cu en s udy a e
in ag eemen wi h he ac ha mixed CE cul u es
like he comme cial CE p oduc B oilac a e su-
pe io in p e en ing Salmonella coloniza ion in
he gas oin es inal ac o b oile chickens. The
signi ican inc ease in he VFA concen a ions,
especially in ha o p opionic acid a 30 d o age,
indica es ha , ei he he s ic ly anae obic bac-
e ia in B oilac emained in he ceca o he es
chickens o B oilac enhanced he es ablishmen
o s ic ly anae obic bac e ia in he ceca o he
es chickens compa ed o he o he ea men s.
CONCLUSIONS AND
APPLICATIONS
1. The esul s o he Salmonella challenge
s udy indica e ha only B oilac o he 3
ea men ma e ials es ed, became es ab-
lished in he gu o he newly ha ched chick-
ens in such a way ha he coloniza ion o
Salmonella was p ohibi ed.
2. The signi ican inc ease in he VFA concen-
a ions, especially ha o p opionic acid a
he end o he ial pe iod, u he indica es
coloniza ion o s ic ly anae obic bac e ia in
he ceca o he B oilac - ea ed chicks.
3. The bi d diges ibili y s udy showed only mi-
no imp o emen s among he di e en ea -
men s which may a leas pa ly depend on
he composi ion o he eed.
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