Efficacy of a Salmonella live vaccine for turkeys in different age groups and antibody response of vaccinated and non-vaccinated turkeys
Hesse et al. BMC Res Notes
Efficacy of a Salmonella live vaccine for turkeys in different age groups and antibody response of vaccinated and non-vaccinated turkeys
Martina Hesse 0
Andreas Stamm 0
Rita Weber 0
Gerhard Glünder 0
0 Clinic for Poultry, University of Veterinary Medicine Hannover , Bünteweg 17, Hannover , Germany
Objective: Human Salmonellosis continues to be one of the most important foodborne zoonoses worldwide, although a decrease in case numbers has been noted in recent years. It is a foodborne zoonotic infection most commonly associated with the consumption of raw egg products but also with meat consumption including the consumption of poultry products. Turkey flocks in Europe have been reported to be affected by Salmonella infection, too. The present study examines the efficacy of a newly licensed Salmonella life vaccine in reducing infections with the Salmonella serovars Typhimurium and Enteritidis in turkeys. Turkeys were vaccinated the first day of life and at the age of 6 and 16 weeks. Groups of birds which had received different numbers of vaccinations were then submitted to challenge trials with either SE or ST. Results: In vaccinated birds Salmonella counts in liver and spleen and, less effectively, in caecum were reduced compared to unvaccinated birds. In several groups serum antibody-titers were statistically significantly higher in vaccinated turkeys than in non-vaccinated ones at day seven post infection, but only in one out of six groups at day 14 post infection.
Salmonella; Turkey; Immunization; Antibody response
Non-host-adapted Salmonellae usually colonize the
digestive tract of turkeys asymptomatically [
Although in case of stress or at a very young age turkeys
may develop severe clinical signs , the most important
problem resulting from Salmonella infections lies in the
transmission to humans. The main source of food-borne
Salmonella outbreaks is the consumption of table eggs
and egg products, but single samples of fresh turkey meat
yielded the highest proportion of Salmonella-positive
]. Control strategies focus on hygiene and
management but also include vaccination [
]. Despite a
recent decrease of the prevalence of human Salmonellosis
in several European countries it is still one of the most
important food borne zoonoses in Europe [
Vaccination of turkeys might help to reduce prevalence in turkey
flocks and transmission to humans further. Barrow et al.
 stress that the use of vaccines has been empirical and
that immunological studies about Salmonella infections
in turkeys are still scarce, although certain aspects of
the humoral immune response have been studied before
]. In two recent studies we examined a bivalent
live Salmonella vaccine for its ability to induce primary
immune reactions after vaccination of 1 day old turkeys
] and for its protective efficacy in turkey poults against
Salmonella challenge infections at the age of 3 weeks
].The latter study found lower Samonella counts in
liver, spleen and caecum of vaccinated turkey poults
compared to unvaccinated poults in challenge trials at
3 weeks of age. No domination of either a TH1-response
or a TH2-response could be determined and no
statistically significant difference regarding the IgG serum
antibody response between vaccinated and non-vaccinated
turkeys after challenge infection was found.
The aim of the present study was to examine the
protective effect of the mentioned vaccine against
Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST)
infections in turkeys in additional age groups and after a
different number of vaccinations. The efficacy was
determined by comparing bacterial counts in caeca, liver and
spleen after challenge. Since it has been shown that
turkeys do not produce antibodies from hatch it should also
be determined if birds which were older than the birds in
our former studies or which were vaccinated more often
would produce a notable serum antibody-response.
Materials and methods
At day of hatch 320 turkeys were housed separately and
divided randomly into two groups of 160 birds each. One
group served as non-vaccinated control group whereas
the other group was directly vaccinated with the
Salmonella live vaccine. Booster immunizations were applied
at the age of 6 and 16 weeks (Table 1). At 2, 6, 16 and
23 weeks of age challenge experiments were conducted
(Table 1). For each challenge experiment 20 vaccinated
and 20 non-vaccinated birds were infected with the
virulent SE strain and 20 vaccinated and 20 non-vaccinated
birds were infected with the virulent ST strain.
At day 7 and 14 post infection 10 individuals per group
were sacrificed by exsanguination after they had been
stupefied by manually applied blunt force trauma and
samples were collected. For vaccinated birds which were
infected with SE at 6 weeks of age only serum samples
d day of life, w week of life
a Challenge with ST or SE, 20 turkeys per each vaccinated or non-vaccinated group
of four birds at 7 and 14 days post infection could be
At the day of hatch commercially available female
fattening turkeys type BUT Big 6 (MoorgutKartzfehn von
Kameke GmbH&Co.KG, Germany) were housed.
Continuous bacteriological and serological monitoring of
the parent flock and of the poults upon arrival were
conducted to ascertain that the birds were free of Salmonella
at that stage of the study. The different groups were kept
separately in isolation units accordant to their
immunization or infection status. Cross contamination between
the immunized group and the control group and between
the four groups in the challenge experiments was
effectively prevented by separate air conditioning, a separate
feeding regime and the change of clothing as well as strict
disinfection of the facilities. Commercial starter feed
and water from the municipal water supply were offered
ad libitum. No antibiotics were added to feed or
drinking water. Water from the municipal water supply in
Germany is suitable to be used as drinking water for humans.
Bacterial strains and culture
The vaccine and the challenge strains used in this study
as well as the preparation of the inocula have been
previously described [
]. Birds were immunized with a
commercial live vaccine (“AviPro Duo”, Elanco Deutschland
GmbH, Bad Homburg). The vaccine contains the
metabolic drift mutant strains Salmonella
Typhimurium-strain ST Nal2/Rif9/Rtt and the Salmonella
Enteritidis-strain SE Sm24/Rif12/Ssq [
]. Each vaccine dose
was prepared to contain 1 × 108 cfu of both strains per
bird which was verified by decimal dilution series. For
infection experiments nalidixic acid resistant mutants
Day post infection
and number examined
of the virulent strains SE K482/91 [
] and STm 27 Nalr
of the phage type DT104 were used. The inocula of the
challenge strains contained 1 × 109 cfu per dose and were
administered with a buttoned cannula directly into the
Samples from caecum ingesta, liver and spleen were
collected during necropsies and used for quantitative
reisolation of the challenge strains as described previously
]. The samples and added phosphate buffered saline
PBS were processed into a homogenous suspension with
an Ultra-Thurrax® (IKA-Werke, Staufen, Germany) with
dispersing tools (Omni-Tip, Omnilab, Bremen). The
caecum ingesta were subjected to a decimal dilution. Two
portions of every dilution step as well as the organ
suspension diluted 1:4 were dispensed on agar plates
selective for the antibiotic resistant challenge strains. The
identification of the SE and ST challenge strains was
confirmed by the affiliation to different serogroups using
Salmonella Test-sera (REF ORND03 and REF ORNH03 by
Dade Behring, Marburg, Germany).
Blood samples of each individual were centrifuged and
the serum stored at − 72 °C. For antibody detection the
ELISA Group B and Group D salmonella Combined
Antibody Test Kit (BioChek, Reeuwijk) was used in
accordance with manufacturer’s instructions. It
discriminates between antibodies directed against LPS antigens
belonging to the Salmonella serogroups B and D.
The absorption of the samples was measured in a
microtitre plate reader at 405 nm wavelength and compared
to the absorption of a positive control. The sample to
positive ratio was determined and the cutoff for positive
values was set at 0.5.
Statistical calculations were conducted with the
computer program SigmaStat®, Version 3.1 (Jandel, Erkrath).
To detect statistically significant differences between
vaccinated and unvaccinated animals t-tests were carried out
if data were normally distributed. If not,
Mann–WhitneyRank sum tests were employed. The significance level was
set at p < 0.05 for all tests.
Results and discussion
The present study tested the effectiveness of a
Salmonella live vaccine in turkeys in different age groups. To
our knowledge this study is the first that assessed the
effectiveness of live vaccination to prevent SE infection in
turkeys over such a long period of time and in so many
different age groups. In the literature we could not find
reports on the use of live vaccines to prevent ST
infections in turkeys.
In challenge experiments vaccinated and
non-vaccinated turkey poults were infected with either a virulent
ST strain or a virulent SE strain. At day seven and 14 post
infection caecum colonization as well as infection of liver
and spleen were evaluated.
One reason for vaccination of domestic poultry is to
reduce Salmonella prevalence in livestock, hence
preventing the contamination of poultry products. A
reduction of Salmonella in the intestine would be important
for this purpose. Additionally, the prevention of systemic
infection allegedly resulting in a diminished colonization
of the reproductive tissues has been named as a goal of
Although it has been argued that Salmonellae in the
cecum lumen are not readily accessible for the humoral
or cell-mediated immunity [
], some studies found
reduced cecal colonization or fecal shedding by the use of
inactivated vaccines in turkeys [
]. Only in some of
our challenge experiments presented here and in a
previously published experiment  cecal colonization was
reduced in vaccinated birds, too (Fig. 1).
At day seven post infection the re-isolation of ST from
22 weeks old vaccinated birds and the re-isolation of SE
from 16 to 22-weeks-old vaccinated animals was
significantly reduced compared with the non-vaccinated
control group. In contrast, 14 dpi generally more colony
forming units of SE were found in the vaccinated than in
the non-vaccinated turkeys with significant differences at
the age of 6 and 16 weeks.
Thus, the tested vaccine does reduce cecum
colonization but not as reliably as desired.
In contrast to findings in the intestine, the systemic
spread of Salmonellae was reduced considerably through
vaccination (Fig. 1). The counts of virulent ST and SE in
livers of vaccinated birds were reduced compared to the
counts in livers of non-vaccinated birds in all age groups
7 days after challenge infection. This could be confirmed
statistically in all groups but one. With the exception of
(See figure on next page.)
Fig. 1 Re-isolation of virulent Salmonella strains from caecum, liver and spleen in different age groups. Figure bars represent the mean log 10
colony forming units/gram from 10 samples of caecum ingesta, liver or spleen, respectively. Error bars represent standard deviation. Statistically
significant differences are indicated by different letters
16 weeks old SE challenged birds; Salmonellae were
completely eliminated from livers of vaccinated turkeys at
day 14 post infection whereas the agent was still present
in livers of non-vaccinated turkeys at a low level at this
From spleens of vaccinated turkeys (Fig. 1)
statistically significantly less ST were isolated than from spleens
of non-vaccinated animals in all age groups at all
timepoints. SE bacterial counts were statistically significantly
lower at day 7 pi in spleens of vaccinated birds which had
been infected at 2 and 22 weeks of age and at day 14 pi in
vaccinated birds which had been infected at 22 weeks of
Thus the vaccine clearly reduced systemic spread in
turkeys, which is in line with previous findings by our
group for 3 weeks old turkeys. In contrast Krüger et al.
] described the failure of a live attenuated vaccine to
protect turkeys against Salmonella infection in a different
setting. Generally studies addressing the success of
vaccination against non-host-specific Salmonella serovars
in poultry have yielded differing and sometimes
conflicting results [
]. For chickens (reviewed by [
]) and ducks
] live vaccines have been reported to confer protection
against Salmonella infection.
In a previous study we could not detect antibody
production of either vaccinated or non-vaccinated turkeys
after challenge at 3 weeks of age. The finding of IgG
antibodies only at a very low level in young turkeys is in
accordance with findings of others [
] who showed that
turkeys started to produce humoral antibodies after
vaccination with an inactivated Bordetella avium vaccine
not before 28 days of age independently from the time of
In the present study higher antibody titers were found
in turkeys which were 6 weeks old at challenge compared
to turkeys examined in our former study, which were
only 3 weeks old at challenge (Fig. 2). We also found that
16- or 22-weeks old animals produced higher titers than
6 week old turkeys.
At day seven post infection vaccinated birds presented
statistically significantly higher antibody titers compared
to the control group with the exception of birds infected
with SE at 6 weeks of age. At day 14 post infection titers
generally had not changed much in vaccinated birds. In
contrast antibody titers in non-vaccinated birds had risen
to roughly the same level as in vaccinated birds or even
higher. At day 14 post infection we could find statistically
significantly higher antibody titers in vaccinated birds
compared to non-vaccinated birds in the groups infected
with ST at 6 weeks of age and SE at 16 weeks of age. For
vaccinated birds infected at 22 weeks of age antibody
titers were statistically significantly lower than in
In summary vaccinated birds produced antibodies
earlier than non-vaccinated birds. High titers of
circulating antibodies have been associated with
protection against systemic infections and could therefore
explain the reduced numbers of colony forming units in
livers and spleens of vaccinated birds [
counts of Salmonellae in internal organs may then
have induced a stronger antibody response in
nonvaccinated turkeys until day 14 post challenge. Similar
results of earlier antibody production in vaccinated
individuals have been reported for Salmonella
vaccination before . However, antibody-production does
not always correlate with Salmonella resistance [
and in the present study we could not find a consistent
relationship between antibody titers and cecum
colonization or infection of internal organs.
For the immune response against bacteria in the gut
lumen IgA antibody titers in the bile would possibly be
even more interesting but were not addressed in this
Due to the experimental design the present study
cannot determine if there is a causal relationship
between antibody response and protection against
Salmonella challenge infections.
TH1-response: T helper type 1 response; TH2-response: T helper type 2
response; SE: Salmonella Enteritidis; ST: Salmonella Typhimuriumdpi day(s)
MH, AS, GG and RW conceived and designed the experiments; MH and AS
performed the experiments; MH, AS, and GG analyzed the data; MH and GG
wrote the paper. All authors read and approved the final manuscript.
The Salmonella challenge strains were kindly supplied by Lohmann Animal
Health Company, Cuxhaven, Germany. The authors would like to thank Sonja
Bernhardt, Sabrina Techel and Katja Stolpe for their excellent support during
the animal experiments. Furthermore, we would like to thank Hilke Bartels for
excellent technical assistance and advice in the laboratory. Additionally, we
would like to thank Lothar Brock for checking the manuscript regarding the
correct use of the English language.
Andreas Stamm received a scholarship from Lohmann Animal Health for work
that was not directly related to the present study. Aside from that the authors
declare to have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The use of animals in this study was reviewed by the animal welfare officer of
the University of Veterinary Medicine Hannover, which includes the scrutiny
of animal welfare, ethics and handling, and then announced to the Lower
Saxony State Office for Consumer Protection and Food Safety according to
§8a(1,2) of the German Animal Health and Welfare Act. Work on this study
was approved under file number 33.9-42502-05-11A153 of the competent
Lohmann Animal Health Company partially funded the bacteriological
examinations. The ELISA was kindly provided by the manufacturer (Biochek). Any
other funding was provided by the Clinic for Poultry, Hannover.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Hafez HM , Jodas S. Salmonella Infections in Turkeys . In: Wray C , Wray A , editors. Salmonella in domestic animals . New York: CABI Publishing; 2000 .
2. Gast RK . Salmonella Infections . In: Saif YM, editor. Diseases of poultry . 1st ed. Ames: Iowa State University Press; 2003 . p. 567 - 8 .
3. European Food Safety Authority . Report of the task force on zoonoses data collection on the analysis of the baseline survey on the prevalence of Salmonella in turkey flocks, Part B . EFSA J . 2008 ; 198 : 1 - 124 .
4. Bundesinstitut für Risikobewertung. Grundlagenstudie zur Erhebung der Prävalenz von Salmonellen in Truthühnerbeständen . 2008 . http://www. bfr.bund.de/cm/208/grundlagenstudie_zur_erhebung_der_praevalenz _von_salmonellen_in_truthuehnerbestaenden. pdf. Accessed 16 Feb 2018 .
5. Immerseel FV , Methner U , Rychlik I , Nagy B , Velge P , Martin G , et al. Vaccination and early protection against non-host-specific Salmonella serotypes in poultry: exploitation of innate immunity and microbial activity . Epidemiol Infect . 2005 ; 133 : 959 - 78 .
6. O 'Brien SJ. The “decline and fall” of nontyphoidal Salmonella in the United Kingdom . Clin Infect Dis . 2013 ; 56 : 705 - 10 .
7. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014 . EFSA J. 2015 ; 13 : 4329 .
8. Robert Koch-Institut . Infektionsepidemiologisches Jahrbuch meldepflichtiger Krankheiten für 2015 . Berlin: Robert Koch-Institut; 2016 .
9. Barrow PA , Jones MA , Smith AL , Wigley P. The long view: salmonella-the last forty years . Avian Pathol . 2012 ; 41 : 413 - 20 .
10. Kremer CJ , O'Meara KM , Layton SL , Hargis BM , Cole K . Evaluation of recombinant Salmonella expressing the flagellar protein fliC for persistence and enhanced antibody response in commercial turkeys . Poult Sci . 2011 ; 90 : 752 - 8 .
11. Thain JA , Baxter-Jones C , Wilding GP , Cullen GA . Serological response of turkey hens to vaccination with Salmonella hadar and its effect on their subsequently challenged embryos and poults . Res Vet Sci . 1984 ; 36 : 320 - 5 .
12. Tenk I , Gyorvary I , Erdei P , Szabo Z , Kostyak A , Matray D. Effect on Salmonella shedding in breeding turkey flocks of vaccine (Salenvac) against Salmonella Enteritidis . Magy. Allatorvosok Lapja . 2000 ; 122 : 737 - 41 .
13. Hesse M , Stamm A , Weber R , Glünder G , Berndt A . Immune response of turkey poults exposed at 1 day of age to either attenuated or wild Salmonella strains . Vet Immunol Immunopathol . 2016 ; 174 : 1 - 10 .
14. Hesse M , Stamm A , Berndt A , Glünder G , Weber R . Immune response to Salmonella infections in vaccinated and non-vaccinated turkeys . Res Vet Sci . 2017 ; 115 : 165 - 73 .
15. Hahn I. A contribution to consumer protection: TAD Salmonella Vac E-A new live vaccine for chickens against salmonella enteriditis . Lohmann Inf . 2000 ; 23 : 29 - 32 .
16. Ludwig HJ , Calsow P . Prevention of Salmonella infections in laying hens by vaccination . Berl Munch Tierarztl Wochenschr . 1992 ; 105 : 96 - 8 .
17. Barrow PA , Methner U . Vaccination against Salmonella infections in food animals: rationale, theoretical basis and practical application . In: Barrow PA , Methner U , editors. Salmonella in domestic animals. 2nd ed. Oxfordshire; 2013 . p. 455 - 75 .
18. Barrow PA , Wallis TS . Vaccination against Salmonella in food animals: rationale, theoretical basis and practical application . In: Wray C , Wray A , editors. Salmonella in domestic animals . New York: CABI Publishing; 2000 .
19. Barrow PA . Immunological control of Salmonella in poultry . In: Blankenship LC , editor. Colonization control of human bacterial enteropathologens in poultry . San Diego: Academic Press; 1991 . p. 199 - 217 .
20. Charles SD , Nagaraja KV , Sivanandan V. A lipid-conjugated immunostimulating complex subunit vaccine against Salmonella infection in turkeys . Avian Dis . 1993 ; 37 : 477 - 84 .
21. Jodas S , Hafez HM . Field investigations on the efficacy of inactivated Salmonella enteritidis vaccine (Salenvac®) in turkey breeder flocks . In: Hafez HM, editor. Proceedings of 4th international symposium on turkey diseases . Berlin; 2002 . p. 259 - 70 .
22. Nagaraja KV , Kim CJ , Pomeroy BS . Prophylactic vaccines for the control and reduction of Salmonella in turkeys . In: Proceedings 92nd annual meet. US Animal Health Association . 1988 . p. 347 - 8 .
23. Krüger A , Redmann T , Krajewski V . Field Investigations on the efficacy of a live vaccine of Salmonella Enteritidis in Meat Turkeys . In: 7th international symposium on turkey diseases . Berlin: German Veterinary Medical Society; 2008 .
24. Shivaprasad HL , Methner U , Barrow PA. Salmonella infections in the domestic fowl . In: Barrow PA , Methner U , editors. Salmonella in domestic animals. 2nd ed. Oxfordshire: CABI Publishing ; 2013 . p. 162 - 92 .
25. Tang T , Gao Q , Barrow P , Wang M , Cheng A , Jia R , et al. Development and evaluation of live attenuated Salmonella vaccines in newly hatched duckings . Vaccine . 2015 ; 33 : 5564 - 71 .
26. Foulman A , Glünder G . Humoral immune response following immunisation of turkey poults with an inactivated Bordetella avium vaccine . In: 3rd International symposium on turkey diseases . Berlin; 2000 .
27. Clifton-Hadley FA , Breslin M , Venables LM . A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimuirum challenge in chickens . Vet Microbiol . 2002 ; 89 : 167 - 79 .
28. Woodward MJ , Gettinby G , Breslin MF , Corkish JD , Houghton S. Theefficacy of Salenvac, a Salmonella enterica subsp. enterica serotype Enteritidisiron-restricted bacterin vaccine, in laying chickens . Avian Pathol . 2002 ; 31 : 383 - 92 .
29. Hassan JO , Mockett AP , Catty D , Barrow PA . Infection and re-infection of chickens with Salmonella Typhimurium: bacteriology and immune responses . Avian Dis . 1991 ; 35 : 809 - 19 .
30. Beal RK , Wigley P , Powers C , Barrow PA , Smith AL . Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge . Vet Immunol Immunopathol . 2006 ; 114 : 84 - 93 .
31. Berthelot-Herault F , Mompart F , Zygmunt MS , Dubray G , Duchet-Suchaux M . Antibody responses in the serum and gut of chicken lines differing in cecal carriage of Salmonella enteritidis . Vet Immunol Immunopathol . 2003 ; 96 : 43 - 52 .
32. Beal RK , Smith AL . Antibody response to Salmonella: its induction and role in protection against avian enteric salmonellosis . Expert Rev Anti Infect Ther . 2007 ; 5 : 873 - 81 .