JCM Accepted Manuscript Posted Online 16 September 2015

J. Clin. Microbiol. doi:10.1128/JCM.01976-15

Copyright 2015, American Society for Microbiology. All Rights Reserved.

One-step identification of five prominent chicken

Salmonella serovars and biotypes

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Chunhong Zhu1,*, Min Yue1*, Shelley Rankin1, Franois-Xavier Weill2, Joachim

Frey3, and Dieter M. Schifferli1#
1

Department of Pathobiology, University of Pennsylvania School of Veterinary

Medicine, Philadelphia, PA, USA
2
Institut Pasteur, Unit des Bactries Pathognes Entriques, Paris, France
3
Institute of Veterinary Bacteriology, Department of Infectious Diseases and
Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Correspondence should be addressed to D.M.S. ([email protected])

Current address: Jiangsu Institute of Poultry Science, Chinese Academy of

Agricultural Sciences, Yangzhou, 225125 Jiangsu, China

Chunhong Zhu and Min Yue contributed equally to this work.

Abstract:
Based on bacterial genomic data, we developed a one-step multiplex PCR assay to
identify Salmonella and simultaneously differentiate the two invasive avian-adapted S.
enterica serovar Gallinarum biotypes Gallinarum and Pullorum, as well as the most
frequent, specific and asymptomatic colonizers of chickens, serovars Enteritidis,
Heidelberg and Kentucky.

Keywords:
Chicken, Salmonella, S. Enteritidis, S. Heidelberg, S. Kentucky, S. Gallinarum, S.
Pullorum, multiplex PCR

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Strains of most Salmonella serovars are zoonotic. Approximately 90% of human

salmonellosis results from ingestion of contaminated food products of animal or plant
origin (1). With over 19,000 reported cases in the US for 2013, Salmonella remains
the most frequently isolated bacterial food pathogen, as determined by the
surveillance network FoodNet which pools the data of 10 US monitoring sites (2). In
parallel to the rise of poultry consumption over the years in the US, the commercial
poultry industry has grown impressively, reaching over 9 billion raised and processed
broilers per year and a yearly production of over 77 billion table eggs, as indicated for
2009 (3). Salmonella is a frequent asymptomatic intestinal colonizer of poultry. Stress
or underlying diseases in young birds create optimal conditions for productive
horizontal transmission of Salmonella sp. Data from the USDA-FSIS suggests that
every fourth raw chicken part is likely contaminated with Salmonella (2). Moreover,
major Salmonella serovars can spread to reproductive organs, leading to vertical
transfer of the bacteria and egg-related salmonellosis (4, 5). Accordingly, poultry and
egg consumption represent a significant source of Salmonella infections in the US.
Four Salmonella serovars are of particular concern to the poultry industry, namely
Enteritidis, Heidelberg, Kentucky and Gallinarum (6). S. Gallinarum is an invasive
agent of chicken salmonellosis resulting in high mortality and morbidity, with biotype
Pullorum (S. Pullorum) which causes "white diarrhea" in young chicken (pullorum
disease), and biotype Gallinarum which is responsible for fowl typhoid (7). Although
this serovar remains endemic in many countries, it has essentially been eradicated
through culling programs in the domestic fowl industry of the USA and several other
developed countries. S. Gallinarum can colonize and/or cause disease in various
domestic and wild birds, which might explain its occasional detection in backyard
birds of developed countries (8). In recent years, S. Enteritidis became a most
frequently isolated serovar in poultry and from foodborne outbreaks linked to poultry
products in developed countries (9). This serovar was suggested to have filled the
ecological niche vacated by the eradicated S. Gallinarum biotypes Pullorum and
Gallinarum (10). Lately, S. Heidelberg has become another major serovar responsible
for foodborne infections from poultry products (11, 12), as well as one of the most
common serovars obtained from non-clinical chicken isolates (9, 13, 14). S. Kentucky
is the most common serotype isolated from chickens and the second most common
one found among retail chicken product in the USA. It has been rarely reported in
human cases in North America (15, 16), although this could change with worldwide
spreading of the ciprofloxacin-resistant ST198 (17).
Here we describe a simple one-step multiplex polymerase chain reaction (PCR)
method to identify major chicken S. enterica subsp. enterica serovars. The approach
was based on designing primers that specifically amplify unique sets of Salmonella
spp. and serovar-associated DNA sequences in one PCR tube (Table 1), taking
advantage of 3,161 available Salmonella genomes, including strains from serovar
Enteritidis (369 genomes), Heidelberg (154), Kentucky (63), Gallinarum (8 biotype
Pullorum and 4 biotype Gallinarum), and 2,563 genomes from 104 other serovars.
2

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The desired specificities were checked by using BLAST (NCBI, non-redundant

nucleotide collection). Strains of the Salmonella genus and Gallinarum biotypes were
identified by primers for differently conserved DNA segments in the their bcf and ste
fimbrial usher genes, respectively (18). Specific primers for serovar Gallinarum
biotype Gallinarum were made by taking advantage of a deletion of 4 nucleotides in
steB of biotype Pullorum. Other specific DNA signatures served as primer targets to
separate serovars Enteritidis, Heidelberg and Kentucky. Briefly, for the multiplex PCR,
pure template DNA (1-5 ng per reaction; MagNA Pure LC DNA Isolation Kit III,
Roche Life Sci., Indianapolis, IN) or crude DNA (approximately 75 ng per reaction,
from bacterial suspensions boiled for 5 min, 107 CFU/l dH2O, using 1 l
supernatants after centrifugation) was amplified with Taq DNA polymerase and a final
concentration of 1.5 mM Mg2+ (Choice Taq Blue, Denville Sci. Inc., South
Plainfield, NJ) using standard protocols. The PCR (25 cycles with an annealing
temperature of 56C) was performed with a Hybaid Thermal cycler (Thermo Fisher
Sci., Waltham, MA). The specificity and compatibility of the primer sets in a
multiplex PCR was assessed using genomic DNA from 128 Salmonella strains that
included a total of 34 different serovars as well as three Escherichia coli and two
Yersinia spp. as negative control strains (Suppl. Table 1).
Agarose gel electrophoresis profiles for each different amplicon sets are visualized
with representative strains in Fig. 1 and the results for all the strains are listed in Table
2. All the Salmonella strains were recognized as such, as were strains of the
Gallinarum biotypes and the Enteritidis, Heidelberg and Kentucky serovars. Thus, the
obtained experimental results were in agreement with the genomic information used
for the primers' design and validated the proposed identification of S. enterica and the
serovar/biotype differentiation among major chicken isolates.
Routine screening of flocks for the presence of Salmonella can be done by
conventional serology which is expensive, as well as time- and labor-consuming.
Based on the restricted number of major serovars found in chicken, extensive
molecular techniques are not always cost-effective, and simpler more focused
approaches could serve as rapid early diagnostic tests. Here, we took advantage of a
small gap in gene steB of biotype Pullorum that was predicted by genomic analysis
(18) to design primers that hybridize to biotype Gallinarum, but not Pullorum DNA,
permitting a one-step PCR differentiation of the two biotypes (Table 2). This method
shortened a previously described two-step technique (19). The addition of primers for
additional chicken-associated serovars all in one multiplex PCR analysis is useful for
the diagnosis of Salmonella in these birds. Although the designed probes are specific
for the identification of serovars Heidelberg, Enteritidis and Gallinarum, serovar
Kentucky shares its PCR profile with serovar Albany, which is not a major chicken
isolate in the USA (13, 14). If needed, these two serovars could be differentiated by a
flagellin-specific PCR (Suppl. Fig. 1). Finally, rarer serovars for which genomic data
are currently unavailable might theoretically share one of the described PCR profiles,
but as such serovars are significantly less frequent in chicken (13, 14), this would be
3

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of minor concern.

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References

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Taken together, this study used (a) genomic sequence data for Salmonella to design a
chicken-specific multiplex PCR diagnostic test and (b) an extensive library of
Salmonella strains and serovars to validate the specificity of the method for the
identification and differentiation of major avian-associated serovars. This simple and
economical test should be useful for specific screening of poultry flocks, particularly
for developing countries or backyard flocks and game birds in developed countries.
Acknowledgements
We thank Leon De Masi for critically reading the manuscript. This work was
supported by China Scholarship Council grant [2013]3018 to CZ, and funds from
NIH grant AI098041, USDA grant 2013-67015-21285 and the PennVet Center for
Host-Microbial Interactions to DMS.

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4

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Table 1. List of primers and concentration used for PCR, with targeted DNA and
amplicons sizes.
Targeted
Final primer
Primers

Sequences (5to 3)

Accession #
Targeted genes

DNA

Amplicon

or loci

(species,

sizes (bp)

concentrations

and

(pmole/ml)

Nt segments
serovars)

bcfC-F

GGGTGGGCGGAAAACTATTTC

bcfC-R

CGGCACGGCGGAATAGAGCAC

AM933172
0.6

heli-F

ACAGCCCGCTGTTTAATGGTG

heli -R

CGCGTAATCGAGTAGTTGCC

steB -F

TGTCGACTGGGACCCGCCCGCCCGC

bcfC

S. enterica

993
25665- 26657

orf (predicted
2

CP005995
Heidelberg

782

helicase)

3226024-3226805

Gallinarum
AM933173
2

steB-R

CCATCTTGTAGCGCACCAT

rhs-F

TCGTTTACGGCATTACACAAGTA

rhs -R

CAAACCCAGAGCCAATCTTATCT

sdf-F

TGTGTTTTATCTGATGCAAGAG

sdf-R

CGTTCTTCTGGTACTTCAGATGAC

gly-F

TTCCAATTGAAACGAGTGCGG

steB

Gallinarum

402
334109-334510
AF370716

sdf locus

Enteritidis

293
4950-5242

orf

"gly"
ABEI01000007

2.6

207
208
209
210

2976016-2976651

AM933173
rhs locus

2.6

636

Gallinarum1

2.6

gly-R

biotype

(hypothetical

Kentucky

170
116981 - 117150

ACTAACCGCTTGGGTTGTTGCTGT

protein)

Absent in biotype Pullorum (Accession number CP006575, locus_tag="I137_00945"), but also present in serovars Enteritidis, Heidelberg,

Kentucky and group 1 serovars, as listed in Table 2.

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Table 2: Bacterial strains used for confirm the specificity of the multiplex PCR assay.
Salmonella enterica serovars and biotypes 1

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Multiplex PCR positive for

bcfC heli steB rhs sdf gly
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

Heidelberg (2)
Enteritidis (11)
Kentucky (4)
Gallinarum biotype Gallinarum (16)
Gallinarum biotype Pullorum (7)
Others: Group 1(68) 2
Others: Group 2 (20) 3
Non Salmonella strains (5)4
1
Numbers of strains in brackets (see Suppl. Table 1)
2
Other S. enterica serovars (group 1) that have the same PCR profile: Paratyphi A (4 isolates),
Paratyphi B var. Java (1), Agona (4), Abortusequi (2), Abortusovis (2), Saintpaul (3), Stanleyville
(1), Typhisuis (2), Braenderup (5), Choleraesuis (24), Ohio (1), Thompson (1), Hadar (2),
Muenchen (2), Newport (6), Berta (2), Dublin (2), Panama (1), Typhi (1), Agoueve (1), Cerro (1).
3
Other S. enterica serovars (group 2) that have the same PCR profile: Schwarzengrund (3).
Typhimurium (2), Bareilly (1), Hartford (1), Montevideo (2), Oranienburg (3), Javiana (6),
Mississippi (1), Pomona (1).
4
Three E. coli and two Yersinia strains.

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Fig. 1. Agarose gel (1.5%) of multiplex PCR amplicons from different bacterial
strains. Representative gel from three comparable experiments. Lanes 1 and 10, 100
bp DNA ladder (NEB, Ipswich, MA); Lane 2, Escherichia coli (DH5a, negative
control); Lane 3, S. enterica group 2, according to Table 2; Lane 4, S. enterica group
1, according to Table 2; Lane 5, S. enterica serovar Enteritidis; Lane 6, S. enterica
serovar Heildelberg; Lane 7, S. enterica serovar Kentucky; Lane 8, S. enterica serovar
Gallinarum biotype Pullorum; Lane 9, S. enterica serovar Gallinarum biotype
Gallinarum.

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