Journal of Applied Microbiology 1998, 84, 125132

Selection of lactobacilli for chicken probiotic adjuncts

M. Garriga, M. Pascual, J.M. Monfort and M. Hugas
IRTA, Meat Technology Center-CeRTA, Granja Camps i Armet, Monells, Spain
6116/02/97: received 19 February 1997, revised 2 May 1997 and accepted 5 May 1997

During inhibitory activity

screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77
strains showed inhibition against enteric indicator strains (Salmonella enteritidis and
Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for
the following attributes: their ability to inhibit all the indicator strains; a high adhesion
efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics,
monensin, bile salts and pH 30. The inhibitory action was not affected by the addition
of catalase and no inhibition was detected after neutralizing the supernatant culture fluid.
The competitiveness of the most promising strains, Lact. salivarius CTC2183 and
CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both
strains were capable of becoming predominant over the indigenous flora in the incubated
chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize
and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum
of the inoculated chicks.
M . G AR R IG A, M . P AS C UA L, J .M . M O NF OR T AN D M . HU GA S . 1998.

INTRODUCTION

The normal intestinal flora is the largest bacterial reservoir

in animals. In healthy animals, the composition of the gut
microflora remains steady, but if the stability is broken, the
pathogenic micro-organisms are able to colonize the gut,
leading to serious infections. Internal factors, such as sudden
changes in the animal itself (Havenaar and Huis int Veld
1992) or external factors such as deprivation of food and
water (Tannock and Savage 1974), trips (Lenener et al. 1984;
Lizko et al. 1984), antibiotic administration and radiation
(Havenaar and Huis int Veld 1992), can break the stability
of the gut microflora of healthy animals.
In chickens, Campylobacter, Escherichia coli and Salmonella
are the main pathogenic micro-organisms that colonize the
intestinal tract. The infection does not cause serious illness in
birds, and production losses are not normally very important.
However, salmonella-infected chickens represent a source of
pathogens for humans, causing severe illness and even death
(Tauxe 1991). In order to prevent these infectious diseases,
live microbial cultures, named probiotics, can be administered to animals to strengthen the barrier function of the
gut microflora and/or for a non-specific enhancement of the
immune system (Sogaard and Suhr-Jessen 1990).
Correspondence to: Dr Margarita Garriga, IRTA, Meat Technology
Center-CeRTA, Granja Camps i Armet, 17121 Monells, Spain
(E-mail: Garriga@monells.irta.es).
1998 The Society for Applied Microbiology

The probiotic bacteria must fulfil the following conditions:

it must be a normal inhabitant of the gut, and it must be able
to adhere to the intestinal epithelium to overcome potential
hurdles, such as the low pH of the stomach, the presence of
bile acids in the intestines, and the competition against other
micro-organisms in the gastro-intestinal tract (Nurmi et al.
1983; Chateau et al. 1993).
Most probiotics contain single or multiple strains of lactic
acid bacteria; they are part of the natural microflora of many
animals, they are generally regarded as safe (GRAS), and
they display antagonistic activity against pathogenic bacteria.
Lactobacilli represent an important component of the intestinal flora in chickens, reaching 109 g1 of the caecal content
(Barnes 1979). The present study was carried out to isolate,
characterize and further select the most suitable lactobacilli
strains for in vivo experiments as chicken probiotic adjuncts.
The competitiveness of the best strains in the chicken gastrointestinal tract has been evaluated.
MATERIALS AND METHODS
Isolation of the bacteria

The crop and/or the intestinal content of 1542 day-old

chicks (50 individuals from three different origins) were
removed, aseptically minced, diluted 1/10 (peptone 01%

126 M . G AR R IG A E T AL .

w/v, NaCl 085% w/v) and vortexed for 30 s. Serial 10-fold

dilutions from the homogenate were made and plated in
Rogosa agar (Difco Laboratories, Detroit, MI, USA). The
incubation was carried out anaerobically for 2 d at 37 C.
After incubation, 296 isolates were randomly sampled and
subcultured on MRS broth (Difco) at 37 C in anaerobic
conditions. The cultures were stored deep-frozen (80 C)
with glycerol (20% v/v) and propagated twice before all the
assays.

sidered positive. At least 10 epithelial cells were examined

per assay.
For examination with a scanning electron microscope
(Zeiss DSM960A, Oberkochen, Germany), samples were
fixed in a solution containing 25% (w/v) glutaraldehyde, 01
mol l1 cacodylate buffer, followed by 1% (w/v) OsO4 in the
same buffer. The samples were processed through a series of
ethanol solutions (50, 70, 90 and 100%), dried in a CO2
Critical Point Drier and coated with gold.

Detection of inhibitory activity

The inhibitory activity was screened by the agar spot test

(Schillinger and Lucke 1989) in MRS agar (Difco), 1% (w/v)
dextrose, under anaerobic conditions at 37 C. The indicator
strains used, Salmonella enteritidis CTC1026 and Escherichia
coli CTC1028, were of animal origin (Table 1). A well diffusion assay with the inhibitory strains was performed (Schillinger and Lucke 1989). The supernatant culture fluid was
tested, neutralized or non-neutralized, and treated with catalase (Sigma, St Louis, MO, USA) at 05 mg ml1 final
concentration.
In vitro adherence assay

The inhibitory strains were tested by the Fuller method

(1973) for their ability to adhere to the epithelial cells of
chicks. Epithelial cells from the crop of chicks aged 12 weeks
were used. The epithelial cell concentration was adjusted to
875 105 cells ml1 and the bacterial cell concentration to
approximately 150 108 cfu ml1.
Bacterial suspension (100 ml) was added to 400 ml of the
epithelial cell suspension and further incubated for 30 min at
37 C in a shaking water bath (20 rev min1). The adhesion
was observed by phase-contrast microscopy (Olympus,
Tokyo, Japan). The bacterial strains showing an adhesion
efficiency of at least 10 bacteria per epithelial cell were con-

Sensitivity of isolates to several parameters

In order to examine the antibiotic sensitivity of the isolates,

MRS broth (Difco) was supplemented with antibiotics
(Sigma) (4 mg ml1 erythromycin, 32 mg ml1 nalidixic acid,
1 mg ml1 streptomycin, 32 mg ml1 chloramphenicol). The
antibiotics were dissolved in water and filter-sterilized; chloramphenicol was dissolved in ethanol. A control containing
an equivalent amount of the solvent was used. The cultures
were incubated anaerobically at 37 C for 48 h and the growth
was assessed as visible turbidity.
Sensitivity of coccidiostats was tested with monensin
(Sigma) dissolved in ethanol and added to MRS broth (05
mg ml1 to 3 mg ml1) and to a moistened commercial feed
mixture (05 mg ml1 to 33 mg ml1).
Resistance to bile salts was tested after growing the isolates
in MRS agar with ox-bile (Oxoid, Unipath Espana, Madrid,
Spain); 1% and 4%, w/v final concentration).
Resistance of the isolates to pH 30 was tested as follows:
overnight cultures of the isolates were centrifuged for 10 min
at 4950 g. After resuspending the pellet in the same volume
of saline solution, it was diluted 1/10 in sterile distilled water
at pH 67 and at pH 30. After 3 h of incubation at 37 C,
the appropriate dilutions were plated in Rogosa agar and
incubated anaerobically at 37 C for 48 h.

Strain
CTC
Serotype
Origin
Others

Salmonella enteritidis
1026
9,12: gm: Poultry litter
NalS, Mot+
Salm. enteritidis
1039
-: gm: Aborted eggs
NalR, MotSalm. enteritidis
1040
9,12: -: Laying hen
NalR, MotSalm. enteritidis
1041
9,12: gm:Poultry litter
NalR, Mot+
Salm. typhimurium
1037
1,19,12: i: 1,2 Rabbit
NalR, Mot+
Salm. typhimurium
1038
1,19,12: i: 1,2 Rabbit
NalR, Mot+
Escherichia coli
1028
O1
*
Type strain
Campylobacter jejuni
1042
Human faeces
NalR, Mot+
Camp. jejuni
1043
Human faeces
NalR, Mot+

*National Institute of Public Health and the Environment (The Netherlands). The strains
were kindly provided by I. Badiola (IRTA-Animal Health Unit).

Table 1 Source and characteristics

of indicator strains

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 125132

P RO BI O TI C L A CT OB A CI LL I 127

Strain identification

The isolates were identified to species level through biochemical tests. Gas production from glucose was anaerobically tested in MRS broth with inverted vials at 37 C for
72 h. Growth at 15 C and 45 C was observed in MRS broth
after 72 h incubation.
The configuration and quantification of lactic acid was
determined enzymically using D and L lactate dehydrogenases
(Boehringer Manheim GmbH Biochemica, Mannheim, Germany). Carbohydrate fermentation was determined using the
API 50CHL system (Baume-les Grottes, France).

to water and feed (commercial mixture). At each sampling

time (15, 22, 41, 65 and 86 h after inoculation), two chickens
were killed by cervical dislocation. The contents of the crop
and caeca were collected, homogenized in 1/10 PBS and
serially diluted before plating in Rogosa agar and in Rogosa
agar with rifampicin (100 mg ml1). The plates were incubated anaerobically at 37 C for 2 d.
The dominance of a given inoculated strain among the
intestinal and crop flora was ascertained by testing rifampicin
resistance and the plasmid profile of a certain number of
rifampicin-resistant colonies (a=005% and b=005%)
according to a progressive colony sampling plan based on the
accumulated binomial distribution (Malegeant 1991).

Plasmid isolation

Lysis and plasmid DNA isolation was carried out according

to the method described by Anderson and McKay (1983).
The agarose (07% w/v in Tris acetate EDTA buffer) gel
electrophoresis was run at 100 V for 2 h in an LKB 2301
Microdrive 1 (Pharmacia Biotech, Uppsala, Sweden).
Aggregation test and coaggregation assay

The aggregation test was performed according to Reniero et

al. (1992). Aggregation was scored positive when clearly visible sand-like particles, formed by the aggregated cells, gravitated to the bottom of the tubes, leaving a clear supernatant
fluid within 2 h.
The coaggregation assay was performed according to Vandevoorde et al. (1992). The coaggregation procedure was
standardized by shaking the cultures for 30 min at 150 rev
min1 and allowing the flocs to settle for 1 h at room temperature before measuring the O.D.. The coaggregation was
expressed according to the Handley et al. (1987) equation.
Inoculation of the chicks

In order to select rifampicin-resistant colonies, overnight

cultures of strains Lact. salivarius CTC2183 and CTC2197
were plated in Rogosa agar supplemented with rifampicin
(100 mg ml1) and incubated anaerobically for 48 h at 37 C.
A single rifampicin-resistant colony from each culture was
selected, subcultured in MRS broth, and confirmed by plasmid profile, inhibitory spectra and aggregation ability. Resistance to rifampicin without selective pressure was tested after
10 passages in an antibiotic-free MRS broth.
The selected rifampicin-resistant strains CTC2183 and
CTC2197 were grown anaerobically in MRS broth for 24 h
at 37 C. The cultures were centrifuged at 4950 g for 10 min
and resuspended in phosphate buffered saline (PBS in g l1,
pH 60: NaCl 80, KH2PO4 034, K2HPO4 121). Twelve, 1day-old chickens of the Label variety were each given 100 ml
of each strain suspension (108 cfu). The birds had free access

RESULTS

The screening of 296 strains of lactic acid bacteria from

the gastro-intestinal tract (crop and intestinal content) of
50 chicks of different origin resulted in 77 strains showing
inhibition against one or more enteric indicator strains (E.
coli, Salm. Enteritidis). Thirty-five strains showed strong inhibition in the agar spot test against both indicator strains, and
adherence to chicken epithelial cells in the in vitro adherence
assay of Fuller (1973). From these strains, 14 were selected
for further assays because of their ability to inhibit all the
indicator strains as well as a high adhesion efficiency (Table
2, Fig. 1). The adhesiveness of these strains was recorded as
positive in the aggregation test of Reniero et al. (1992) (Table
2); they were also able to inhibit other salmonella strains of
animal origin, such as Salm. typhimurium CTC1037,
CTC1038, Salm. enteritidis CTC1039, CTC1040 and
CTC1041, and Campylobacter jejuni CTC1042 and CTC1043
of human origin, in the agar spot test.
The inhibitory effect was not affected by the addition of
catalase to the supernatant culture fluid; no inhibition was
detected after neutralizing the fluid.
The selected strains were resistant to pH 30, bile salts
(4%) (except for CTC2233, which was discarded), resistant
to 8 mg ml1 erythromycin, 32 mg ml1 nalidixic acid, 1 mg
ml1 streptomycin, 32 mg ml1 chloramphenicol (except for
CTC2183 and CTC2244), and sensitive to 4 mg ml1 ampicillin.
After the biochemical and physico-chemical assays, the
strains were identified as Lactobacillus salivarius because of
their ability to grow at 45C, no gas production from glucose,
production of only L-lactic acid, and by the fermentation
pattern of the API test. Plasmid profiles were used in order to
discard identical strains (Fig. 2). Five strains showed plasmid
profiles identical to other strains, and the following eight
different strains were retained: CTC2049, CTC2112,
CTC2166, CTC2176, CTC2183, CTC2186, CTC2197, and
CTC2244.
The coaggregation results of paired strains is shown in

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 125132

128 M . G AR R IG A E T AL .

Escherichia
Salmonella
Strain
coli
enteritidis
Adhesion
Aggregation
CTC
Origin
CTC1028
CTC1026
efficiency*
time**

2023
gic
++
++
1
nd
2049
crop
++
++
2
60
2051
crop
++
++
2
120
2053
crop
++
++
1
nd
2055
crop
++
++
2
60
2057
crop
++
++
1
nd
2071
crop
++
++
1
nd
2102
crop
++
++
1
nd
2112
crop
++
++
2
15
2146
crop
++
++
2
15
2166
crop
++
++
2
15
2176
crop
++
++
2
15
2178
crop
++
++
1
nd
2182
crop
++
+++
1
nd
2183
crop
++
+++
2
15
2185
crop
++
+++
1
nd
2186
crop
++
++
2
15
2188
crop
++
+++
1
nd
2193
crop
++
++
1
nd
2197
crop
++
++
3
15
2213
crop
++
++
1
nd
2231
crop
++
++
2
15
2233
crop
++
++
2
nd
2243
crop
++
++
1
nd
2244
crop
++
++
3
15
2253
gic
++
++
1
nd
2255
gic
++
++
1
nd
2260
gic
++
++
2
15
2262
gic
++
++
1
nd
2269
gic
++
++
1
nd
2283
gic
++
++
1
nd
2288
crop
++
++
1
nd
2290
crop
++
++
1
nd
2291
crop
++
++
1
nd
2293
crop
++
++
1
nd

+, Inhibition zone 5 mm; ++, 10 mm; +++, 15 mm.

*1, At least 10 bacteria/epithelial cell; 2, 30; 370.
**, In minutes.
nd=Not determined; gic=gastro-intestinal content.

Table 3, where coaggregation is expressed as the mean relative

O.D. reduction compared with the unpaired strains. In particular, Lact. salivarius CTC2049 and CTC2197 were most
frequently involved in coaggregation reactions and were also
positive when paired with Salm. enteritidis CTC1026 (data
not shown).
As monensin is the most commonly used coccidiostat in
chicken production, sensitivity to monensin was tested in all
the selected strains; Lact. salivarius CTC2183 was the most
resistant strain (2 mg ml1) when assayed in MRS broth.

Table 2 Inhibition of indicator

strains by chicken lactobacilli

When strains CTC2183 and CTC2197 were grown in commercial chicken feed mixture moistened and supplemented
with monensin, the strains showed higher resistance than in
MRS broth (up to 9 mg ml1). The assayed strains CTC2183
and CTC2197 were capable of predominating over the
endogenous flora in the incubated chicken feed mixture as
detected by their plasmid profile (data not shown).
Lact. salivarius CTC2183 and CTC2197, inhibitors and
highly adherent and resistant to monensin, bile salts and pH
30, were chosen for the inoculation of 1 day-old chicks.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 125132

P RO BI O TI C L A CT OB A CI LL I 129

Fig. 1 Scanning electron micrograph of a

crop epithelial cell with Lactobacillus

salivarius CTC2197 attached

colonies, according to the sampling plan, it was determined

that Lact. salivarius CTC2197 was more competitive than
CTC2183.
DISCUSSION

Fig. 2 Plasmid profiles (lanes 114) from Escherichia coli V517

(Macrina et al. 1978), Lactobacillus salivarius CTC2049,

CTC2051, CTC2055, CTC2112, CTC2146, CTC2166,
CTC2176, CTC2183, CTC2186, CTC2197, CTC2231, CTC2244
and CTC2260

Resistance to rifampicin was maintained after 10 passages in

MRS broth without selective pressure. This trait allowed
differentiation between the inoculated micro-organisms from
indigenous strains. Throughout the trial (4 d), both rifampicin-resistant strains dominated over the sensitive lactobacilli in the crop and caeca of inoculated chickens (Table
4). By checking the plasmid profiles of rifampicin-resistant

Nurmi and Rantala (1973) were the first to report that oral
dosing with gut content containing native gut microflora from
salmonella-free adult birds could protect the chicks from
infection by Salmonella spp. however, this practice could
result in the widespread transmission of any undesirable bacteria. For that reason, attempts have been made to develop
defined bacterial mixtures for commercial use (Mead and
Impey 1987).
As host specificity of bacterial strains is well recognized
and documented (Fuller 1975; Suegara et al. 1975; Barrow et
al. 1980), isolation of lactobacilli from chickens was undertaken in this study as a first step towards developing a chicken
probiotic adjunct. The crop was chosen as a source of strains
because its microflora, consisting mainly of lactobacilli (Sarra
et al. 1992), provides an effective barrier against colonization
by human foodborne pathogens.
Among 296 isolates, eight strains were selected because of
their good biological properties as successful probiotics, their
moderate or strong adherence ability, their resistance to a
number of antibiotics and coccidiostats, bile salts and pH 30,
and their antimicrobial spectrum against enteric bacteria. The
adherence capability to intestinal cells and/or mucus would
be advantageous for these strains as it gives them a great

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 125132

130 M . G AR R IG A E T AL .

CTC
2049
2112
2166
2176
2183
2197
2231
2244

2049
0
17
57
0
144
31
0
33
2112
0
0
0
0
19
15
0
2166
0
61
0
0
44
112
2176
0
12
26
0
0
2183
0
19
56
0
2197
0
39
0
2231
0
0
2244
0

Values are the average of three experiments.

Table 3 Percentage of coaggregation

Sampling
Crop
CTC2183 CTC2197 Caeca
CTC2183 CTC2197
time**
*
(%)
(%)
*
(%)
(%)

15
516
0
100
790
25
75
22
558
75
25
753
25
75
41
637
25
75
856
50
50
65
529
0
100
892
0
100
86
706
0
100
820
17
83

*Counts are expressed in log cfu g1 and are the mean of two samples.
**Hours after challenging Lactobacillus salivarius CTC2183 and CTC2197.

Table 4 Rifampicin-resistant lactobacilli

ecological advantage (Sarra et al. 1992); this is, presumably,

a prerequisite for colonization (Havenaar and Huis int Veld
1992), Rada et al. (1995) reported the colonization of the
digestive tract of newly-hatched chickens with Lact. salivarius
51R despite the fact that its adhesion to epithelial cells was
negligible. Additionally, some strains gave a good score in
coaggregation assays between intestinal lactic acid bacteria.
Lactobacillus salivarius CTC2049, CTC2166, CTC2183 and
CTC2197 also coaggregated with pathogenic bacteria (Salmonella CTC1026), indicating that the inhibition of enteric
bacteria could be mediated in physical ways as previously
reported by Reid et al. (1988). These authors suggested that
the dominance of inhibitor-producing lactobacilli on the urogenital epithelium of humans, and the ability of these organisms to interact closely with uropathogens such as E. coli,
would constitute an important host defense mechanism
against infection. The prevalence of coaggregation among
lactobacilli supports the idea that intrageneric interactions
might be of considerable ecological significance, increasing
the chances for the establishment of bacteria at a low residence
time of colonization as suggested by Vandevoorde et al.
(1992).
The inhibitory activity displayed by these strains, which is
another characteristic that could enhance their competitiveness in the gastro-intestinal tract, remained

between paired strains of

chicken lactobacilli

from the crop and caeca of inoculated

chicks

unchanged when the culture supernatant fluids were treated

with catalase, showing that the inhibition was not related to
hydrogen peroxide. No inhibitory zones were observed when
the culture supernatant fluids were adjusted to pH 65; this
suggested that an acidic compound other than lactic acid, or
in addition to it, could be responsible for the inhibition as
the addition of the same amount of lactic acid as that produced
in the culture supernatant fluid was unable to produce the
same inhibition (data not shown). Jin et al. (1996) reported
that the inhibition of different Salmonella and E. coli Serotypes by 12 lactobacilli strains isolated from chicken intestine
was presumably related to organic acids.
In general, the lactobacilli strains isolated in this study
showed a higher resistance to antibiotics such as erythromycin
and streptomycin than the chicken intestinal lactobacilli isolated by Rada et al. (1991), although the same authors found
more resistant strains to ampicillin that were obtained in this
study.
Chicken diets often contain antimicrobial compounds used
for the control and treatment of infectious diseases. This is a
matter of concern because the over-use of antibiotics in meat
leads to the development of antibiotic-resistant bacteria in
domestic animals. In the future, it would be necessary to
verify the genetic localization of the resistance to avoid health
risks for consumers.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 125132

P RO BI O TI C L A CT OB A CI LL I 131

Ionophores such as monensin are used extensively in the

poultry industry as they are efficient coccidiostats, but they
also inhibit the growth of lactobacilli and other Gram-positive
bacteria by dissipating the cation and proton transmembrane
gradients as reported by Pressman and Fahim (1982). In this
study, the minimum inhibitory concentration of monensin
against poultry lactobacilli in MRS broth varied from 05 mg
ml1 to 2 mg ml1, which agrees with the results obtained by
Marounek and Rada (1995) with three strains of Lact. salivarius isolated from chickens. These authors also proved
that resistance to ionophores in their natural environment is
higher than in laboratory media, as observed in this study
when the growth of Lact. salivarius strains CTC2183 and
CTC2197 was assessed in chicken feed mixture. The bacterial
glycocalyx, which is synthesized in a natural environment
with solid particles (Costerton et al. 1981) and protects cells
from the action of antimicrobials, could be lost in laboratory
cultures, meaning that the MICs of ionophores found in
liquid media cannot be extrapolated (Marounek and Rada
1995).
When the competitiveness of the most promising strains
was assayed in vivo, it was concluded that Lact. salivarius
CTC2197 was the best strain for further experiments because
it was able to colonize and overcome the indigenous flora, as
well as the other putative probiotic strain, in the crop and the
caeca of the chickens.
New studies for evaluating the ability of this strain to
compete successfully with the indigenous bacteria, and to
minimize enteric colonization in poultry, are in progress in
which chicks are being challenged with Salmonella and the
probiotic strain.

ACKNOWLEDGEMENTS

The authors are grateful to Yolanda Beltran for her technical

assistance and to Pere Plana for his collaboration in handling
the animals. This work was supported by the Comision Interministerial de Ciencia y Tecnologa (CICYT), project
AGF94-0220. M. Pascual is the recipient of a CIRIT predoctoral grant.

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