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Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Development and evaluation of live attenuated Salmonella vaccines in

newly hatched duckings

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Tian Tang a,b,1 , Qun Gao a,b,1 , Paul Barrow d , Mingshu Wang a,b,c, , Anchun Cheng a,b,c ,
Renyong Jia a,b,c , Dekang Zhu b,c , Shun Chen a,b,c , Mafeng Liu a,b,c , Kunfeng Sun a,b,c ,
Qiao Yang a,b,c , Xiaoyue Chen b,c
a

Avian Diseases Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Yaan, Sichuan 625014, PR China
Key Laboratory of Animal Diseases and Human Health of Sichuan Province, Yaan, Sichuan 625014, PR China
c
Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, Sichuan 611130, PR China
d
School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
b

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a r t i c l e

i n f o

a b s t r a c t

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Article history:
Received 4 May 2015
Received in revised form 28 August 2015
Accepted 4 September 2015
Available online xxx

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Keywords:
Duck
Salmonella
Vaccine

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1. Introduction

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Domestic ducks remain a major source of zoonotic Salmonella enterica infections for man worldwide and
approaches to protection should include vaccine-mediated immunity. With this in mind we developed
several genetically dened mutants in a virulent duck Salmonella typhimurium isolate TT-1. From initial
tests for virulence in day-old ducks, rpoS, hilA, and slyA mutants retained some virulence so were
not studied further. Amongst the mutants showing greater attenuation, ssrB, phoPQ, ompR, and
clpP also showed high levels of protection when 1-day-old ducks, which were vaccinated orally, were
challenged 1 week later demonstrating the capacity to protect ducks in the rst few weeks of life when
they are most susceptible and when the risk of infection is greatest. Immunized ducks triggered Ompspecic IgG, IgM, and IgA responses and raised IL-2 and IFN- levels in the serum coupled with IL-4
suppression.
2015 Published by Elsevier Ltd.

Although duck rearing for meat and eggs is a relatively minor

component of the poultry industry in western countries, duck meat
has traditionally been a major source of animal protein in many
Asian countries. World production was approximately 4.4 million
tons in 2013 and may exceed 4.5 million tons in 2015. Production increased by 40% between 2000 and 2010, greater than the
increase in production of other poultry species with the corresponding increases in Asia and particularly China being 44% and
47% respectively. The estimated production in China is 2.76 million tons representing 82% or production in Asia (www.fao.org;
www.thepoultrysite.com).

Corresponding author at: Key Laboratory of Animal Disease and Human Health of
Sichuan Province & Avian Disease Research Center, College of Veterinary Medicine,
Sichuan Agricultural University, 46# Xinkang Road, Yucheng District, Yaan 625014,
PR China. Tel.: +86 835 2885774; fax: +86 835 2885774.
E-mail address: mshwang@163.com (M. Wang).
1
These authors contributed equally to this study and should be listed as rst
authors.

Food-borne zoonotic pathogens are thus of increasing interest to

the duck industry, some of which, such as Salmonella, may additionally cause considerable economic losses [1]. Isolation of Salmonella
serovars from ducks has been recorded from many countries and
recently from Egypt [2], South Korea [3], Malaya [4], Vietnam [5]
and less recently from the United Kingdom [6]. Isolation has been
reported from 22 of the 28 Chinese provinces and regions since
1981 [713] with isolation from raw duck meat reaching 69% in
some cases [14]. Given the general absence of hygienic controls
associated with duck rearing it is not surprising that a variety of
serovars are frequently isolated [2,15].
Compared with the available information on Salmonella infection in domestic fowl our knowledge of the course of infection in
ducks is much poorer [16]. This paucity of information includes
approaches to control for a species of poultry where hygienic limitations are much greater than with chickens. The possibilities of
using competitive exclusion ora have not been explored extensively although live vaccines administered orally can induce a
similar effect by virtue of their ability to colonize the intestine
[17]. Antibiotic therapy results in resistance in Salmonella; levels
of resistance are variable but can be high with a tendency toward
multi-resistance [2,18] in some cases with strains resistant to 16 or
more antimicrobials [3,10,13,15].

http://dx.doi.org/10.1016/j.vaccine.2015.09.004
0264-410X/ 2015 Published by Elsevier Ltd.

Please cite this article in press as: Tang T, et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched
duckings. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.09.004

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Vaccination has been tested in ducks and although S. Enteritidis aroA mutant was found not to be protective the immunity
generated by infection with a wild-type S. Enteritidis indicates
that vaccination should be possible and clearly a practical option.
There are unpublished reports of effective protection using commercially available vaccines developed for use with chickens (see
[16]). In chickens live attenuated Salmonella vaccines generally confer better protection than killed vaccines [19], because the former
stimulate both cell-mediated and humoral immunity [20]. To date,
many genetically dened live attenuated Salmonella vaccines have
been reported for use with chickens but not assessed for their efcacy in ducks.
Auxotrophic strains, especially those conferred by mutations in
the aromatic pathway [21,22] or in purine biosynthesis [23,24],
are attenuated and effective live vaccines. Mutants with defective
virulence determinants may also be considered as candidate vaccines. These include a key Salmonella Pathogenicity Island 1 (SPI1)
regulator hilA mutation which reduces invasive ability and virulence when inoculated intragastrically [25,26] and defective SPI2
mutants such as ssrB [27,28]. In addition, null mutations in genes
expressing global virulence regulators such as PhoP/PhoQ, OmpR,
SlyA, ClpP, RpoS and Fis also induce signicant reductions in virulence [2939]. Some of these mutations have been found to be
attenuating and protective in chickens [40]. Of the mutations mentioned above none has been tested thus far for their attenuating
effects in ducks and neither has their potential protective ability.
We have produced several genetically dened mutants of wild
type strain TT-1, a highly virulent S. typhimurium strain isolated
from the carcass of a young duck in Henan province, China. Their
attenuation and safety, immunogenicity and protection against a
lethal challenge were assessed in young ducks.

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2. Materials and methods

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2.1. Bacterial strains, media, and standard genetic manipulations

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All bacterial strains used in this study are listed in Table 1. Strains
TH4702 (LT2 pKD46) and TH6701 (LT2araBAD925::tetRA) were
kind gifts from Prof. Kelly T. Hughes at the University of Utah. Cells
were routinely grown in LuriaBertani (LB) broth. Antibiotics were
added to LB agar or broth at the following nal concentrations:

100 g/ml ampicillin for PKD46-containing strains and tetracycline

at 15 g/ml. The generalized transducing phage of P22HT105/1 int201 was used in all transductional crosses [19,41].
2.2. Construction of tetRA insertion mutants and markless
deletions
To generate strain TT-2 (TT-1 pKD46), P22 phage lysates of
TH4702 were used to transduce pKD46 [42] into TT-1, selecting
for ampicillin resistance.
The construction of tetRA insertion mutants and markerless deletions followed protocols described previously [43]. The
resulting insertion (TT-1 to TT-9) and deletion (TT-11 to TT16) derivatives are shown in Table 1. Strain TH6701 was used
as template for PCR amplifying tetRA. All primers are listed in
supplementary material.
2.3. Measurement of in vitro growth kinetics

S. typhimurium
TH4702
TH6701
TT-1
TT-2
TT-3
TT-4
TT-5
TT-6
TT-7
TT-8
TT-9
TT-10
TT-11
TT-12
TT-13
TT-14
TT-15
T-16

Genotype

LT2 pKD46
LT2 araBAD925::tetRA
Wild-type
TT-1 pKD46
TT-1 hilA::tetRA
TT-1 ssrB::tetRA
TT-1 phoPQ::tetRA
TT-1 ompR::tetRA
TT-1 rpoS::tetRA
TT-1 slyA::tetRA
TT-1 clpP::tetRA
TT-1 hilA
TT-1 ssrB
TT-1 phoPQ
TT-1 ompR
TT-1 rpoS
TT-1 slyA
TT-1 clpP

Source

Hughes KT
Hughes KT
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study

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To evaluate the in vitro growth rate of mutant strains, overnight

broth cultures were diluted to an OD600 of 0.05 using fresh trypticase soy broth (TSB) and incubated at 37 C at 180 rev/min. At
hourly intervals 1 ml samples were taken and the OD measured.
The experiments were done in triplicate.
2.4. Virulence assay for Salmonella TT-1 and isogenic derivatives
1-Day-old Sichuan ducks were purchased from a local commercial hatchery, and housed in cages. Prior to the experiment, all
birds were screened for Salmonella maternal antibody (SMA) using
an indirect ELISA described below and only SMA-free birds were
used. To determine the 50% lethal dose (LD50 ) of the parent strain
TT-1 in 1-day-old ducks, 50 birds were divided randomly into 5
groups, and dosed orally with 0.2 ml aliquots of Salmonella TT-1
ranging from 106 CFU to 1010 CFU. The control group was dosed
with the same volume of diluent (phosphate-buffered saline containing 0.01% gelatin, BSG, [44]). The method of Reed and Muench
method was used to calculate the LD50 [45]. To evaluate the virulence of isogenic derivatives, the oral inoculation dose of each
mutant was adjusted to the concentration of 10-fold and 100-fold
the LD50 value for TT-1. The number of birds which died or were

Table 1
Bacterial strains, virulence and protection.
Strain

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Virulence of wild type and mutant

strains under different doses (No. of
survived/total)

Protection (No. of survived/total) after

different vaccinating doses

1.2 109 CFU

1.2 1010 CFU

1.2 106 CFU

1.2 108 CFU

/
/
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/
7/10
10/10
10/10
10/10
5/10
9/10
10/10

/
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0/10
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/
/
/
4/10
10/10
10/10
10/10
1/10
6/10
10/10

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/
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/
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/
20/20
20/20
20/20
/
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20/20
Non-immunized birds

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/
/
/
/
/
/
/
17/20
20/20
20/20
/
/
16/20
8/20

/Means
not tested.

Please cite this article in press as: Tang T, et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched
duckings. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.09.004

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killed humanely during a period of 2 weeks from the infection were

recorded.

To determine the colonization ability of mutant strains in vivo,

ve groups of 30 1-day-old ducks were dosed orally with 108 CFU
of the ssrB::tetRA, ompR::tetRA, phoPQ::tetRA, clpP::tetRA
mutants (rather than the antibiotic sensitive derivatives) or the
wild type strain TT-1 in 0.2 ml BSG. One similar sized group of ducklings was dosed with the same volume of BSG. Three birds per group
were killed on days 1, 3, 5, 7, 14, 28 and 42 post-infection (pi). Liver,
spleen and caeca were removed aseptically, weighed and homogenized with 1 ml of sterile PBS using an IKA Disperser (IKA, Staufen,
Germany). Aliquots of 100 l of decimal dilutions of the resulting
homogenates were plated on MacConkey agar supplemented with
or without 15 g/ml of tetracycline. Five colonies from each bird
were selected randomly to conrm genotypic markers by PCR.
2.6. Immunization and challenge
2.6.1. Experiment 1
Five groups of 36 1-day-old ducks were administrated orally
with 1.2 108 CFU (in 0.2 ml BSG) of a suspension of the ssrB,
ompR, phoPQ, or clpP mutants respectively together with an
non-immunized control group. Non-immunized birds were inoculated orally with 0.2 ml of BSG diluent. At 1, 3, 5, 7, 14, 21, 28,
35, 42, 49, and 56 days post-immunization (dpi) three birds per
group were killed humanely for serum and bile analysis by ELISA
and serum bactericidal assay (SBA). Samples were stored at 70 C
until tested.
2.6.2. Experiment 2
Nine groups of 20 1-day-old ducks were inoculated orally with
1.2 106 CFU or 1.2 108 CFU (in 0.2 ml BSG) suspension of the
ssrB, ompR, phoPQ, or clpP, respectively together with an
non-immunized control groups dosed with the same volume of
BSG. 1 week after the primary immunization, birds from all groups
were challenged with 1.2 1010 CFU of the wild type strain TT-1.
Mortality was recorded for 2 weeks as before. The short interval
between immunization and challenge was chosen because most
ducks normally become infected in the rst 2 weeks of life.
2.7. ELISA and serum bactericidal assay

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2.8. Statistical analysis

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3. Results

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3.1. Oral LD50 of parent strain TT-1 and the isogenic derivatives

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2.5. Colonization of mutants of in vivo

The levels of interferon gamma (IFN-), interleukin-2 (IL2) and IL-4 in duck sera were measured using commercial
ELISA kits (Novateinbio, Boston, USA). Extraction of outer membrane proteins (OMPs) from Salmonella TT-1 was performed as
described previously [46]. Goat anti-duck IgG (KPL, Gaithersburg,
USA), IgM (Antibodies-online, Shanghai, China) and rabbit antiduck IgA (Antibodies-online, Shanghai, China) conjugated with
horseradish peroxidase (HRP) were used to determine the titers of
serum-specic IgG, IgM and biliary IgA against Salmonella OMPs,
respectively. Color development (absorbance) was recorded at
450 nm using a Bio-Rad model 680 microplate reader. The SBA was
performed as reported previously [47].

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Statistical analysis was performed using IBM SPSS Statistics software (IBM, NY, US). The differences of bacterial counts, cytokine
concentration, in vitro growth rate and antibody titers were evaluated with One-Way ANOVA and Tukeys tests between groups.
Signicance was taken as a P value of <0.05.

To investigate the virulence of the parent strain TT-1, we determined the LD50 for 1-day-old ducks. Following infection, all birds
dosed with 3.8 109 or 3.8 1010 CFU succumbed to infection
within a 14-day observation period, whereas inoculation with
3.8 108 , 3.8 107 or 3.8 106 CFU resulted in 70%, 30%, or 0%
mortality, respectively. The LD50 of TT-1 in 1-day-old duckings was
calculated as 1.2 108 CFU.
Next, we sought to attenuate the virulence of S. typhimurium
TT-1 to generate vaccine candidates. To this end, we constructed
several genetically dened mutant strains in TT-1 and evaluated
their virulence in 1-day-old ducks. Inoculation of groups of 10
birds with 1.2 109 cells of rpoS, hilA or slyA mutants of TT-1
resulted in the deaths of 5, 3, and 1 birds, respectively. Inoculation with 1.2 1010 cells of rpoS, hilA, or slyA mutants of TT-1
resulted in the deaths of 9, 5, and 4 birds, respectively. All birds
inoculated with 1.2 109 or 1.2 1010 cells CFU of the phoPQ,
clpP, ompR, or ssrB mutants survived. Inoculation of ducks
with 1.2 1010 CFU of the TT-1 parental strains resulted in all birds
dying. The results are shown in Table 1.
3.2. The growth kinetics of mutant strains in vitro
Amongst the four most attenuated vaccine candidates (phoPQ,
ompR, clpP, and ssrB), strain TT-16 (clpP) exhibited a signicant growth defect in comparison with the other mutants and
parent strain TT-1 (P < 0.01). In contrast, the ompR (TT-13) mutant
showed a greater growth rate than all other strains tested (P < 0.05)
(Fig. 1).
3.3. The colonization of mutant strains in target organs
To assess the colonization of the four candidate vaccine strains
in vivo, we used the tetracycline resistant precursors of ssrB (TT11), phoPQ (TT-12), ompR (TT-13), and clpP (TT-16), namely
ssrB::tetRA (TT-4), phoPQ::tetRA (TT-5), ompR::tetRA (TT-6),
and clpP::tetRA (TT-9) to monitor their dynamics in the liver,
spleen and caeca of vaccinated ducks. The results are shown in
Fig. 2AC. Both clpP::tetRA (TT-9) and ssrB::tetRA (TT-4) colonized the liver and spleen efciently after inoculation with ca.
108 CFU bacteria. In comparison, the phoPQ mutant was not recovered at all from either liver or spleen during the experiment. The
ompR mutant persisted for a shorter period of time being eliminated

Fig. 1. Growth curves for mutants in TSB over a 4 h period. Warmed TSB inoculated
with a 24 h culture to OD600 0.05. At hourly intervals, 1 ml samples were taken
and the OD measured. The experiment was repeated three times. Differences analysis between groups were determined using one-way ANOVA and Tukeys tests (**,
P < 0.05; ***, P < 0.01). P < 0.05 was considered as signicant difference.

Please cite this article in press as: Tang T, et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched
duckings. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.09.004

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Fig. 2. Colonization of duck liver, spleen, and caeca by attenuated mutants. AC: Mean values of Log 10 viable numbers per gram of liver, spleen, and caeca from three birds,
respectively. Differences analysis between groups at each time point determined using one-way ANOVA and Tukeys tests. P < 0.05 was considered as signicant difference.

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by 14 days post-inoculation. All test mutants were recovered from

caeca during the rst 4 weeks of infection. With the exception of the
ssrB mutant, the clpP, phoPQ, and ompR mutant strains were eliminated from the caeca by day 42 pi. The colonization data of the wild
type strain TT-1 was only recorded for the rst 3 timepoints, since
half of the birds had died by 7 days post-infection.

immune bile at 14 dpi. Thereafter, the IgA titer increased gradually

and reached a peak on day 35 and persisted in bile for 56 days.
The results of serum bactericidal assay indicated that the sera
produced by the ompR mutant exhibited a greater bactericidal
activity than immune sera that were collected from birds vaccinated with the phoPQ, clpP, or ssrB mutants (P < 0.05) (Fig. 4).

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3.4. Salmonella specic antibodies induced by vaccine candidates

3.5. Cytokine response to attenuated mutant strains

We investigated the antibody responses induced by the phoPQ

(TT-12), ompR (TT-13), clpP (TT-16), and ssrB (TT-11) mutant
strains. All vaccinated strains elicited robust IgM, IgG and IgA antibody responses using TT-1 OMPs as detecting antigen (Fig. 3AC).
The OMP-specic IgM was rst detectable in immune sera collected at 5 dpi with the titer increasing progressively and peaking
on day 28 pi, followed by a gradual decline (Fig. 3A). The ssrB
mutant induced signicantly higher levels of OMP-specic IgM
than that induced by the phoPQ and ompR mutants at 28 dpi
(P < 0.05). The OMP-specic serum IgG was rst detected at 7 dpi.
Birds immunized with the ssrB or clpP mutant produced the
highest OMP-specic IgG titers. The IgG titers declined before those
of IgM and in some cases were not detectable at day 49 pi when the
experiment ended (Fig. 3B). The anti-OMP IgA initially appeared in

As results shown in Fig. 5AC, Levels of IL-2 in sera collected

from birds vaccinated with the ompR, clpP, and ssrB mutants
increased progressively from day 3 to day 7 pi and then decreased.
The highest concentrations of IL-2 were induced by the clpP
mutant strain whereas the phoPQ mutant induced no signicant
levels of IL-2 at all (Fig. 5A). The levels of IFN- in immune sera
increased from day 5 pi and peaked on day 7 pi (Fig. 5B). The concentrations of IL-4 measured in response to vaccination with all strains
were reduced and were almost a mirror image of the IFN- prole.
On day 5 pi, the levels of IL-4 in antisera, collected from immunized birds, were below the detection limit (Fig. 5C). By day 42 pi,
the concentrations of IFN- and IL-4 in immune sera had returned
to the normal day 0 level and showed no statistically signicant
differences to the control birds.

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Please cite this article in press as: Tang T, et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched
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Fig. 3. Titers of serum OMP-specic IgM (A), IgG (B), and bile OMP-specic IgA (C) following vaccination. To determine the titer of IgM and IgG, the initial dilution of serum
were set at 1:80 and the negative value was set at 1:40. For measurement of IgA, the initial dilution of serum was set at 1:20 and negative value was set as 1:10. Differences
analysis between groups were determined using one-way ANOVA and Tukeys tests. P < 0.05 was considered as signicant difference.

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3.6. Protective efcacy of attenuated Salmonella mutants

4. Discussion

Following the assessments for virulence and persistence above,

the phoPQ (TT-12), ompR (TT-13), clpP (TT-16), and ssrB (TT11) mutants were evaluated as live vaccines against challenge with
the virulent parent strain TT-1. As shown in Table 1, ducks vaccinated orally with 1.2 106 or 1.2 108 CFU of phoPQ or ompR
mutant strain showed complete protection against lethal challenge.
Groups of birds immunized with 1.2 108 cells of clpP or ssrB
mutant strains showed partial protection (16/20 and 17/20 survival, respectively). In contrast, only 8/20 non-immunized ducks
had survived by 2 weeks.

We have produced several attenuated mutants of a virulent S.

typhimurium strains isolated from diseased ducks and assessed several of these for virulence, pathogenicity and for their ability to
induce immune protection in ducks.
The strain TT-1 had an LD50 of 108 CFU for 1-day-old ducks. This
is clearly very high compared with LD50 values for 1-day-old chickens where values of 101 104 [48] are more common depending on
strain but are comparable to other similar studies involving different serovars including S. typhimurium [17]. This suggests that
several commercial lines of duck are currently inherently relatively

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Fig. 4. Viability of mutant strains incubated in ltered duck serum. 10 l of parental

strain TT-1 (108 CFU/ml) were mixed with 90 l of pooled sera that collected from
vaccinated ducks at 28 days post-infection. One-way ANOVA and Tukeys tests were
used for determine the differences between groups (**, P < 0.05; ***, P < 0.01). P
value < 0.05 was considered as signicant difference.

resistant to systemic salmonellosis although this situation may

have changed from previous decades where experimental mortality has been produced [49,50].
After oral inoculation the ssrB, phoPQ, clpB, ompR and, to slightly
lesser extent, the rpoS mutants showed a considerable degree of
attenuation and the rst four were studied further. The hilA and
slyA were discarded as showing too little attenuation.
Salmonella pathogenicity island 1 (SPI1) is closely involved in the
invasion of enterocytes. As the key regulator, HilA directly affects
the expression of SPI1 genes. Inactivation of hilA gene resulted in a
moderate attenuation in mice via oral route [51,52]. In this study,
we observed a similar attenuation of our hilA (TT-10) mutant in
1-day-old ducks, which caused a >10-fold increased LD50 compared
to the wild type strain TT-1.
In contrast to past ndings indicating that a S. typhimurium rpoS
null mutant was avirulent in mice [39], our investigation suggested
that the rpos (TT-14) mutant strain retained some virulence in
newly hatched ducks. This result, in combined with previous report

Fig. 5. The measurement of IL-2 (A), IFN- (B), and IL-4 (C) in duck sera at 1, 3, 5, 7, 14, 21, 28, 35, 42, and 49 days post-inoculation. Difference analysis between groups was
done using one-way ANOVA and Tukeys tests. P < 0.05 was considered as signicant difference.

Please cite this article in press as: Tang T, et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched
duckings. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.09.004

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that rpoS mutant was highly pathogenic for young chicks [53], suggested that inactivation of the rpoS gene was unable to effectively
reduce the pathogenesis of Salmonella in birds. In addition to the
rpos strain, the incomplete attenuation of slyA (TT-15) mutant
was also not anticipated since a similar construct in S. typhimurium
strain was fully attenuated in mice [54]. However, the difference in
virulence shows that extrapolation from mammals to birds may
be inappropriate and can lead to spurious assertions as found
previously [55]. In addition, 1-day-old birds are immunologically
immature and possess a very rudimentary intestinal microbial ora
which may thus contribute to increased colonization and virulence
by pathogenic bacteria.
Although mutagenesis of clpP or ssrB reduced virulence in
ducks, it did not completely eliminate invasiveness since both the
clpP::tetRA (TT-9) or SsrB::tetRA (TT-4) strains were isolated
from both liver and spleen. In contrast, inactivation of phoPQ or
ompR dramatically diminished the ability of S. typhimurium to colonize the internal organs. Previous ndings indicated that a clpP
null mutant was hyper-agellated [56], and resulted in a chronic
infection in mice [57] whereas our investigation suggested that the
clpP::tetRA strains were cleared from the liver and spleen within
3 weeks post-infection. This may be attributable to the signicant
growth defect of our clpP mutant demonstrated in vitro. This has
not been observed previously where growth defects have only been
seen at reduced temperature or in the presence of higher salt concentrations [58,59]. The relative persistence of the ssrB::tetRA
strain was unexpected, as a ssrB mutant of S. typhimurium was
entirely unable to colonize the liver and spleen in mice [60] and
again indicates differences between mammals (mice) and avian
species.
A strong level of immunity was triggered by the phoPQ (TT-12),
ompR (TT-13), clpP (TT-16), and ssrB (TT-11) mutants against
challenge by the parent strain 1 week after vaccination. This is
clearly a very short period between vaccination and challenge and
not sufcient for a full T cell-mediated immunity to be stimulated
to generate the degree of protection observed. Circulating Ompspecic IgG and IgM were detected by 1 week post-vaccination but
the highest titers were not detected until a full 4 weeks after vaccination and would have been unlikely to be central to the protection
observed. It is well known that in mammals the presence of live
bacteria in the tissues stimulates a high degree of protection mediated by components of the innate immune system [61,62], likely
to include macrophages and polymorphonuclear neutrophil leukocytes (PMN). A similar degree of protection is also seen in chickens
soon after vaccination with live, attenuated vaccines [20]. It seems
likely therefore that a major component of the resistance observed
was related to this inducible non-specic immunity and it would
have been interesting to investigate the protective effect against
serologically unrelated Salmonella serovars.
In addition to the humoral immune response, we were also
interested in stimulation of cell-mediated immunity induced by
the vaccine candidates, since such immunity is closely involved in
the immune clearance of Salmonella [20,63]. As a potent T lymphocyte stimulating factor, IL-2 is a major cytokine produced during
the primary response of Th cells. Upon differentiation into Th1
and Th2 effector cells, IL-2 production declines and is replaced by
production of Th1-like (IFN-) or Th2-like (IL-4) cytokines [64]. In
the present data we found that the increased production of IL-2 in
immune sera of ducks were initially detected at day 3 pi, which indicating that Th cells were primed and were involved in the immune
response at this time. By the following 2 days (day 5 pi), IFN- levels
had increased and persisted for 35 days. This was associated with
a reduction of IL-4. This combination suggested that the vaccines
had begun to stimulate a Th-1 cell mediated immune response.
In addition to early protection primarily involving immune
cells, a competitive exclusion effect induced by colonization of

the intestine of chickens [40] and ducks [17] by live, attenuated

Salmonella vaccines could have contribute to some of the protection seen although this is thought to be largely a physiological
phenomenon associated with extensive colonization rather than a
specic immunological effect. However, whatever the basis of the
protection observed, it is practically important to protect young
ducks during the rst few weeks of life when most infection occurs
and when their susceptibility is greatest.
Contribution
TT and AC designed the experiment. WM, DZ, RJ, SC, QY, XC and
KS guided the experiment. TT and QG performed the experiments.
TT and PB wrote this paper.
Acknowledgement
This research was supported by China Agricultural Research Q4
System (CARS-438), National Science and Technology Support
Program for Agriculture (2015BAD12B05/2011BAD34B03) and
Innovative Research Team Program in Education Department of
Sichuan Province (12TD005/2013TD0015).

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Conict of interest statement: No conicts of interest.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.vaccine.2015.09.
004.
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