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Previous Article | Next Article 
Applied and Environmental Microbiology, January 1999, p. 260-263, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Epidemiology of Campylobacter
jejuni in Broiler Flocks Using Randomly Amplified
Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in
Its Transmission
Randy E.
Payne,1
Margie D.
Lee,2
David W.
Dreesen,2 and
Harold
M.
Barnhart1,*
Department of Environmental
Health1 and
Department of Medical
Microbiology,2 College of Veterinary
Medicine, University of Georgia, Athens, Georgia 30602-2102
Received 18 June 1998/Accepted 6 October 1998
 |
ABSTRACT |
Poultry has long been cited as a reservoir for
Campylobacter spp., and litter has been implicated as a
vehicle in their transmission. Chicks were raised on litter removed
from a broiler house positive for Campylobacter jejuni.
Litter was removed from the house on days 0, 3, and 9 after birds were
removed for slaughter. Chicks were raised on these three litters under
controlled conditions in flocks of 25. None of these birds yielded
C. jejuni in their cecal droppings through 7 weeks. Two
successive flocks from the same Campylobacter-positive
broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were
determined for each flock. Randomly amplified polymorphic DNA
(RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates.
A high prevalence (60%) of C. jejuni in flock 1 coincided
with the presence of an RAPD profile not appearing in flock 2, which
had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was
used to determine if strains with different RAPD profiles and different
prevalence rates contained different 23S sequences. RAPD profiles
detected with higher prevalence rates contained a spacer in the 23S
rRNA region 100% of the time, while RAPD profiles found with lower
prevalence rates contained an intervening sequence less than 2% of the
time. Data suggest varying colonizing potentials of different RAPD
profiles and a source other than previously used litter as a means of
transmission of C. jejuni. These molecular typing methods
demonstrate their usefulness, when used together, in this epidemiologic investigation.
| |
INTRODUCTION |
The mode of transmission of
Campylobacter jejuni into broiler flocks has been an issue
for investigation in recent years. Although the literature supports
litter as a possible means of transmission of C. jejuni to
susceptible chicks under controlled conditions (9), the role
of litter as a medium for the maintenance of the organism and its
subsequent transmission to later flocks has not been fully evaluated.
Hatcheries have been implicated in the introduction of
Salmonella spp. to broiler flocks (2). However,
because of the suggested viable but nonculturable characteristics of
C. jejuni (4, 13), such a connection between
breeder flocks and C. jejuni colonization of broiler flocks
has proven elusive to date. Furthermore, this type of vertical
transmission is doubted by many researchers (1, 16). Recent
advances in molecular microbiology have resulted in genetic typing
methods that lend themselves to epidemiological studies. The ability to
trace organisms from environmental sources to broiler flocks by using
genetic information may prove to be a powerful investigative tool in
determining the mode of transmission of a given organism. Randomly
amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR were used to
establish genetic profiles of C. jejuni isolates cultured
during this study. Genetic typing of recovered C. jejuni
isolates was used to trace strains of the organism through successive
flocks to pursue the epidemiologic objectives of this study. One
objective of the investigation was to determine if previously used
litter in a poultry house acts as a reservoir for C. jejuni
under experimental conditions. The study also investigated the role of
used litter in the transmission of C. jejuni to successive
flocks in the same poultry house.
| |
MATERIALS AND METHODS |
Litter study.
A commercial broiler flock found to be
positive for C. jejuni (flock 1) and the successive flock
placed in the same house (flock 2) were used in this study. On days 0, 3, and 9 after the first flock was taken to slaughter, used litter was
removed and transferred to PDRC (Poultry Disease Research Center,
University of Georgia, Athens, Ga.), a controlled study site. Newly
hatched chicks obtained from the area commercial hatchery used to
supply flocks 1 and 2 were placed on the used litter at the site. The controlled house contained no physical barriers between individual pens
except for wire. All pens used for the study were separated by at least
one empty pen. The pens, approximately 5 by 10 ft, were disinfected
prior to the introduction of chicks, and 2 to 3 in. of the collected
used litter was placed in each pen.
The study began on the day flock 1 was taken to slaughter, designated
day 0 of the experiment. Twenty-five chicks were placed in a pen on day
0 litter. In a separate pen, 25 chicks were placed on fresh wood
shavings as a control group. An additional 25 chicks were sacrificed
for evidence of C. jejuni in their ceca. On day 3, a second
load of litter was transferred from the poultry house to the controlled
site and 25 more hatchlings were placed on the used litter. On day 9, the final load of litter was transferred to the controlled site and 25 hatchlings were again obtained and placed on this litter. Each group
was raised for 49 days. Biosecurity measures, such as wearing of
sterilized plastic boot covers and smocks, were used when pens were
entered. No antibiotics were administered to the birds with the
exception of feed containing bacitracin, which is widely used
throughout the industry as a growth promoter. Fresh, chlorinated tap
water was supplied to the pens daily, and all birds in the study were
fed in a manner consistent with common industry standards.
Sample collection.
Cecal droppings were sampled on a weekly
basis during the rearing of the two successive commercial broiler
flocks. Grow-out time for each flock was 49 days. The first samples
were taken from each flock during the 3rd week of rearing, and the last
samples were taken on the 7th week. This sampling protocol was the same for both farm flocks and for the controlled flock located at PDRC. Freshly deposited cecal droppings were collected with a sterile swab,
placed in a 17- by 100-mm snap-cap culture tube containing 9 ml of
0.1% sterile buffered peptone water, and placed on ice for transport
to the laboratory. Sample collection was random but covered the entire
length and breadth of the broiler house. Forty samples were collected
on each sampling day for the farm broiler house flocks (flocks 1 and
2). Samples were pooled, two per pool, for a total of 20 samples for
laboratory analysis. Ten samples were collected from the controlled
flocks on each sampling day, and samples were not pooled.
Laboratory analysis.
Cecal droppings were placed in 1-quart
Ziploc bags containing 100 ml of Hunt's enrichment broth
(5) with antibiotics and supplements, with recovery and
culturing of the organism according to the U.S. Department of
Agriculture method (12).
Isolates confirmed as C. jejuni were stored at 
70°C on
Microbank beads (ProLab Diagnostics, Austin, Tex.). Single colonies were picked from C. jejuni growth plates and placed on beads
per the manufacturer's instructions. Isolates were recultured on
brucella-FBP agar for genetic typing procedures.
Genetic typing.
DNA template was made by the whole-cell
technique for gram-negative organisms proposed by Woods et al.
(18). A hot air thermocycler (Rapidcycler; Idaho Technology,
Idaho Falls, Idaho) was used to amplify the DNA.
RAPD-PCR.
RAPD-PCR typing was performed with the OPA 11 primer, 5'-CAATCGCCGT-3', with 4 mM MgCl2
(5). Cycling parameters for RAPD-PCR consisted of three
cycles. The first cycle was comprised of two cycles with a denaturing
step at 92°C for 30 s, an annealing step at 36°C for 7 s,
and an extension step at 27°C for 70 s, with a slope of 1.0. This cycle had two repetitions. The second cycle shared the same
temperatures as the first cycle, but the denaturing step had a duration
of 1 s, the annealing step had a duration of 7 s, and the
extension step had a duration of 60 s. The second cycle used a
slope of 6.0 and had 38 repetitions. A holding cycle of 72°C for 4 min followed the first two cycles and ended the amplification process.
23S rRNA-PCR.
23S rRNA-PCR typing was performed with 2 mM
MgCl2, forward primer 5'-TCGGCGGAAAATATAACGGGGCTA-3',
and reverse primer 5'-CTCAACTTAATTATCGCTACTCAT-3', according to the method of Trust et al. (15).
Amplification of 23S rRNA consisted of two cycles. The first cycle was
a holding step at 96°C for 5 min, with no repetitions. The second
cycle was a denaturation step at 94°C for 10 s, an annealing
step at 50°C for 10 s, and an elongation step at 72°C for
35 s, with a slope of 2.0 and 30 repetitions.
After amplification, reaction tube contents were placed in wells cut in
1.5% agarose gels and electrophoresis was conducted at 80 V for 60 min. After electrophoresis, gels were stained with ethidium bromide
(Sigma, St. Louis, Mo.). A 100-bp ladder standard (Boehringer Mannheim,
Indianapolis, Ind.) was used to measure DNA banding patterns. DNA
banding profiles were viewed with a UV light source and photographed.
| |
RESULTS |
Prevalence.
Prevalence rates for C. jejuni
colonization were calculated as percentages of positive cecal
droppings. None of the groups in the controlled flock reared on
previously used litter yielded C. jejuni in their cecal
droppings, including the control group and cecal samples taken from
day-old hatchlings. Neither flock 1 nor flock 2 yielded
Campylobacter isolates prior to the 4th week of sampling.
Flock 1 yielded the higher prevalence rate for C. jejuni
throughout the study except during the 5th week of sampling, when flock
2 experienced an increase in prevalence. At the time of harvest, flock
1 yielded a 60% prevalence rate for C. jejuni colonization
while flock 2 yielded only 28% prevalence at harvest time, as shown in
Fig. 1. All quality assurance parameters
during isolation and identification were satisfied for data accuracy and consistency, and percentages derived from sampling are believed to
be reflective of actual C. jejuni colonization profiles for each flock sampled.
RAPD-PCR typing.
Campylobacter isolates recovered from
flocks 1 and 2 were grouped by profiles obtained by RAPD-PCR. Groupings
were made by observation of banding patterns throughout RAPD-PCR runs.
RAPD types with amplicons of 1.5 kb were denoted group A. Subgroups of
group A were identified as types having amplicons that appeared in
addition to the 1.5-kb band. Group A1 consisted of isolates that had a
1.5-kb band only. Group A2 organisms had a 1.3-kb band that appeared
with the 1.5-kb band, and group A3 organisms were Campylobacter sp., with a 1.1-kb band that appeared in
addition to the 1.3- and 1.5-kb bands. Group A4 organisms comprised a
group that had only a 1.1- and a 1.5-kb band. Group B organisms, on the
other hand, did not express a 1.5-kb band but expressed a banding
pattern consisting of 0.6-, 0.7-, and 1.0-kb amplicons. Bands less than
0.6 kb were not used for grouping purposes because their presence was
inconsistent and not reproducible. The difficulty with these minor
bands has been substantiated by others (10). Banding
patterns observed were consistent and repeatable among triplicate
RAPD-PCR experiments. Bands other than those specified above were
observed, but only those amplicons mentioned were used for grouping.
Figure 2 illustrates these RAPD profiles.
A total of 67 isolates from flock 1 were examined, of which 63 (94%)
produced an RAPD product. Fifty-one of these products (81%) were
identified as subgroups of group A, which occurred throughout the
sampling period from the 4th to the 7th week. Of these 51 isolates, 10 isolates (19%) were placed in group A1, 20 (39%) were placed in group
A2, and 21 (41%) were placed in group A3. The remaining 12 isolates
(23%), which were characterized as group B, were found only during the
7th week of sampling, immediately preceding the harvest of flock 1. However, group B isolates constituted 54% of the isolates recovered
that week.
Only 28 isolates were recovered from flock 2. Of these, 27 (96%)
yielded RAPD products. All patterns observed were placed in group A. Twenty (74%) of these isolates yielded products characteristic of
group A1, while the remaining seven isolates (26%) were identified as
group A4. No group B isolates were identified in flock 2. The occurrences of group A isolates, as determined by the RAPD-PCR method,
are presented in Table 1.
23S rRNA typing.
The presence or absence of an intervening
sequence (7) in the 23S genome of the isolates was
determined to detect differences in parentage between groupings based
on RAPD results. Isolates having a 167-bp product do not have a
nontranscribed spacer region in the 23S gene. Isolates that yield a
310-bp product have the spacer. Figure 3
illustrates 167- and 310-bp products and their absence or presence
within the 23S rRNA region.
Of the 52 isolates recovered from flock 1 that were grouped into RAPD
group A (inclusive to all subgroups of group A), all but one yielded
167-bp amplicons. However, of isolates identified as group B, 12 (100%) yielded 310-bp amplicons. Only 1 (1.9%) of the 51 group A
isolates contained the 23S spacer. In flock 2, 16 of the 27 group A
isolates were examined with 23S rRNA-PCR. None of these isolates
contained the spacer.
| |
DISCUSSION |
The role of litter.
One objective of this study was to further
investigate the role of litter in the transmission of C. jejuni to subsequent flocks reared in the same broiler house
environment. Because no chickens raised in a controlled environment on
previously used litter were positive for C. jejuni, the
evidence in this study does not support an association with previously
used litter and the maintenance or transmission of C. jejuni
to successive flocks, nor does it support an earlier conclusion by
Willis and Murray (17) that broilers raised on used litter
become colonized. The limited data presented here suggest that C. jejuni is not transmitted from the hatchery to the controlled
flock. This supports evidence reported by Berndtson et al.
(3) in an epidemiological study in which parent flocks did
not seem to transmit the organism to farm flocks.
Genetic typing.
All isolates obtained from flock 1 and flock 2 were genotyped by the RAPD-PCR and 23S rRNA-PCR techniques for
identification of genetic profiles. Band patterns produced by RAPD-PCR
were grouped in terms of similar patterns developed on agarose gel
electrophoresis. A comparison of genetic information acquired with the
use of these procedures was made to determine if isolates belonging to
certain RAPD-PCR groups originated from a common source, or a common
parental line, by the presence or absence of a spacer within the 23S
rRNA genome. Genomic identification methods using the 23S rRNA sequence have potential as epidemiological tools because of the conserved nature
of 23S genes. As reported by Ludwig and Schleifer (8), there
seems to be greater discrimination potential with 23S rRNA genes than
with 16S rRNA genes. Campylobacter has been shown to have
three loci of 23S genes in its genome (11). Also, the
literature presents no evidence of there being some 23S loci which do
not contain the spacer present with loci which do. Our data supports this assumption, since none of the strains produced multiple products in 23S rRNA-PCR.
Campylobacter isolates with RAPD group A profiles appeared
throughout flock 1 and flock 2, suggesting a maintenance of RAPD group
A organisms through successive flocks. It seems plausible that C. jejuni group A types may be maintained from a common source.
Our study determined that C. jejuni colonization prevalence
was much greater at the time of harvest of flock 1 than of flock 2, with a second RAPD profile appearing only in flock 1. Stern et al.
(14), during a study of competitive exclusion on cecal colonization of chicks, observed that different isolates of C. jejuni exhibited different potentials for colonization, possibly caused by differences in phenotypic expression between strains. Group B
RAPD profiles appeared only in the 7th week of sampling in flock 1 but
comprised 54% of all recovered C. jejuni isolates for that
sampling period. This suggests that this group of isolates may have a
greater ability to colonize than do group A isolates, which were common
to both flock 1 and flock 2. The presence of the 23S spacer in the
genome of isolates in RAPD group B demonstrates that these isolates are
of a different parental line and that they may have been introduced
from a source internal or external to the broiler house environment but
one not common to the source of group A.
Our study suggests that while the RAPD-PCR method is useful in
epidemiologic investigations, it may be too sensitive when used alone
to group C. jejuni isolates, because differences found in
the RAPD group A data were not confirmed by the 23S rRNA-PCR method.
The 23S rRNA method did not differentiate between subgroups A1, A2, A3,
and A4. Therefore, it may be that these isolates are of the same
parental lineage and that the differences expressed in the RAPD
patterns may be evidence of genetic recombination that occurred in the
organism through the grow-out periods of flock 1 and flock 2. It seems
evident that when RAPD-PCR methods are used, they should be
complemented by a PCR method, such as the 23S rRNA-PCR method, that
examines a more conserved region of the genome. Our RAPD-PCR (group B)
data and its 100% correlation to the presence of spacer regions
demonstrate that the 23S rRNA-PCR method is complementary to the
RAPD-PCR method.
Our data suggest that the common source of Campylobacter
isolates was not previously used litter. C. jejuni is
present in a variety of types of samples in a broiler house
environment, such as water, litter, and dust, etc. Application of the
appropriate PCR techniques would appear to be useful not only in the
investigation of Campylobacter in other areas of the broiler
house but also in other applications in the poultry industry.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the U.S. Poultry and Egg
Association and the U.S. Department of Agriculture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Environmental Health, 206 Environmental Health Building, University of Georgia, Athens, GA 30602-2102. Phone: (706) 542-2454. Fax: (706) 542-7472. E-mail: hbarnhar{at}arches.uga.edu.
| |
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Applied and Environmental Microbiology, January 1999, p. 260-263, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
