259
FIELD INVESTIGATIONS ON THE EFFICACY OF
INACTIVATED
SALMONELLA ENTERITIDIS VACCINE (SALENVAC®)
IN TURKEY BREEDER FLOCKS
1Silvia Jodas and 2H.M. Hafez
1Poultry Health
Service, Schaflandstr. 3/3, 70736 Fellbach, Germany
2Institute of
Poultry Diseases, Free University Berlin Koserstr.21, 14195 Berlin,
Germany
Summary
The present study
was carried out to evaluate the efficacy of a Salmonella
Enteritidis (SE)
inactivated vaccine under field condition in a turkey breeder
farm with Salmonella Enteritidis history.
On turkey breeder
farm with multiple age systems (two flocks in production and
two in brooding), Salmonella Enteritidis (SE) was isolated. Firstly SE
was
isolated from
flock no. 4 in the brooding house at 6th week of age. Two weeks
later SE was
detected in faecal samples from layer flock no. 1 at 53rd week of
age. This flock
was slaughtered immediately. Six months later SE was detected
in flock 3 at 51st
week
of age. This flock was also slaughtered immediately.
Flock 2 remained
Salmonella negative during lifetime.
In aim to control
the infection several hygienic measures, treatment as well as
vaccination trials
were carried out. Flock 4 was vaccinated once at 26th week of
age with a killed Salmonella Enteritidis vaccine intramuscularly
(Salenvac.,
Intervet), which
is currently, not licensed for turkey in Germany. Flock 5 was
vaccinated twice
at 16th and 26th week of age. All following
restocked (flocks 6,
7 and 8) were
vaccinated 3 times at week 7, 16 and 26. Salmonella shedding in
vaccinated flocks
was investigated by intensive bacteriological examination
from faecal
samples, cloacal swabs, hatching eggs, meconium, hatchery fluff
and one-day-old
poults collected at several intervals. In addition, the serological
response was
determined serologically using commercial available SE ELISAtest
(IDEXX).
After introducing
of the vaccine at the farm no Salmonella
spp. could be isolated
neither from
faecal samples of turkey breeder flocks nor from samples collected
at the hatchery.
The application of the killed vaccine 3 times intramuscular
results in a
better serological response in turkeys. The results indicate, that the
application of
inactivated vaccine together with improvement in the production
hygiene has been
remarkably successful in control Salmonella
Enteritidis
infection.
260
Introduction
Salmonellosis and
salmonella infections in turkey are distributed worldwide and
result in severe
economic losses when no effort is made to control them (Hafez
and Jodas, 2000).
The large economic losses are caused by high poults
mortality during
the first four weeks of age, and poor poults quality. Furthermore
Salmonellas are
responsible for reductions in egg production in breeder flocks,
high medication
costs, and high costs for eradication and control measures.
Members of the
genus Salmonella pose a serious threat to the food-animal
industry. The most
important aspect, of Salmonellas is the continuing effect of
salmonella
contaminated turkey meat and meat-products on public health.
Therefore eradication
and control of salmonella infections is necessary to
reduce the
vertical and horizontal transmission.
The protection of
human health against diseases and infections directly or
indirectly
transmissible from animals to man (zoonosis) is of paramount
importance in the
European Union. In order to establish systems for monitoring
of certain
zoonoses and to create control towards Salmonella in animals the
Council Directive
92/117/EEC concerning measures for protection against
specified zoonoses
and specified zoonotic agents in animals and products of
animal origin in
order to prevent outbreaks of food-borne infections and
intoxications has
been adopted. However the Council Directive 92/117 dose not
considers turkeys.
Therefore currently no governmental regulation on
salmonella
infections in the EU includes turkeys. Also in case of positive testing,
no legislations or
obligations exist at present. This Council Directive has to be
repealed by a new
Directive and a new Regulation. Now, the Commission has
prepared
proposals, which are currently in discussion and they include also
monitoring and
control of Salmonella in turkeys
(January 2002.).
The objective of
the Directive is to ensure sufficient
monitoring of zoonoses
and agents thereof
in the Member States and in the Community to collect
necessary
information for the evaluation of trends and sources of zoonoses and
agents thereof.
This evaluation shall support the action to be taken to prevent
and control
zoonoses and agents thereof in Member States, at Community level
and in third
countries.
The objective of
the present study is to evaluate the efficacy of a Salmonella
Enteritidis inactivated vaccine under
field condition in a turkey breeder farm with
Salmonella Enteritidis history.
261
Materials and Methods
Structure of the breeder turkey farm
The vaccination
trials were carried out on a breeder turkey farm with multiple
age system.
Breeder brooder, laying flocks and male flocks are kept on the
same farm. The
farm consist of three houses. House 1 is divided into three
unites. Unites 1
and 2 used as brooding unites for two different flocks. The third
unite is used to
keep male turkeys for semen collection for artificial insemination
of layer flocks 1
and 2. Two layer flocks are mostly in production and kept in two
production houses
(house 2 and 3). Each of both brooding flocks has 2800
female and 300
male birds. At week 18 and after selection female and male
birds are divided
and reared in separate unites. At the end of brooding at week
28, the female
turkeys were transferred to the laying house, where they kept till
the end of the
production period. Each layer flock has about 2300 females and
each tom flock
consist of about 180 males. The hatching eggs were fumigated
on the farm
immediately after collection and then delivered to the hatchery. The
hatchery and the
parent farm belong to the same owner.
Samples
In the hatchery
meconium, fluff, hatching eggs environmental swabs and day
old poults, were
taken at different intervals and examined bacteriologically.
On the breeder
farm faecal and blood samples were collected at different
intervals from
several flocks and examined bacteriologically and serologically. In
addition,
Bacteriological examination for detection of
salmonella
The examinations
were carried out according to the method described by Hafez
et al. (1993) and
Aleksic et al (1994) with slight modification. Samples were preenriched
in Buffered
Peptone Water (1:10) and incubated at 37°C for 24 hours.
0.1 ml of this
pre-enriched broth was transferred to 9.9 ml Rappaport Vassiliadis
(RV) enrichment
broth (1:100) and incubated at 41.5°C for 48 hours. Then the
RV broth’s were
streaked on Brilliant Green Phenol Red Agar (BGA) and
Rambach plates and
incubated at 37°C for 24 hours. Salmonella suspected
colonies were
identified serologically using slide agglutination tests.
Serological examination
Commercially
available ELISA test kit SE - ELISA (H:g,m) was used (IDEXX,
Maine, USA). The
ELISA testing procedure and the interpretation of the results
were performed
according to the instructions of the manufacturers. The used
serum dilution was
1:2.
Vaccine
Salmonella Enteritidis inactivated vaccine (Salenvac®, Intervet) was
administered
intramuscular with a dose of 0.5 ml per bird. The vaccine contain 1
x 104,5
cfu/dose.
The
vaccine is currently, not licensed for turkeys in Germany.
262
SE-Vaccination trials under field conditions
Start of
vaccination was in July 1999. Totally 5 flocks (No. 4, 5, 6, 7, 8) were
vaccinated.
Vaccination scheme is shown in table 1.
Table 1:
Vaccination scheme
Results
Results of bacteriological and serological
investigations before starting of
the vaccination
In March 1999 Salmonella Enteritidis PT4 (SE) was isolated in
the hatchery
from routinely
taken meconium samples.
In year 1999 SE was isolated from 7 out of 231 examined meconium
samples
from the hatchery.
In addition SE was detected in 10 out of 112 tested fluff
samples, 2 out of
70 investigated eggs and in 6 day old poults out of 306 tested.
From 146
environmental swabs collected at hatchery Salmonella Enteritidis
could not be
isolated (Table 2).
Table 2: Results
of bacteriological examination in hatchery in year 1999
Immediately after
the first SE isolation all
parent flocks were intensively
bacteriologically
examined in aim to determine the source of the infection.
On the breeder
farm firstly in May 1999 SE was isolated from
faecal samples
from laying parent
flock No.1 and from a 6 week old brooding flock (flock No. 4)
Between June and
November 1999 no Salmonellas were isolated neither from
samples taken in
the hatchery nor from samples taken from the parent flocks. In
December 1999 Salmonella enteritidis was detected in faecal
samples of non
vaccinated parent
layer flock no. 3 and in the hatchery from fluff and meconium
samples. (Fig. 1).
263
Figure 1: Time
scheme of the SE isolation at the
turkey parent farm and at
the hatchery
In flock no. 1 SE was isolated one time at 53 week of age
immediately before
slaughter. In the
subsequent taken serum samples 45% had antibodies against
SE. In flock no. 2 SE was not isolated during live time. The taken serum
samples had no
antibodies against SE (Table 3).
In flock no. 3 SE
was isolated with 51 and 53 week of age immediately before
slaughter (Table
4). The examined serum samples in week 25, 27, 40 and 47
reacted
serological negative.
Jan Mar May Jul Sep Okt Dez Jan Jan Sep
99 99 00 01 01
Flock 1 Flock 3
SE - isolation from parent flocks: Faecal samples
Flock 4
SE - isolation at the hatchery: meconium, fluff,
eggs
264
Table 3: Result of
bacteriological and serological investigation of flock 1
and 2
Flock 1 Flock 2
Week of
live
Faecal
samples
Serum
samples
Week of
age
Faecal
samples
Serum
samples
5 neg n.d. 2 neg
n.d.
7 neg n.d. 13 neg
n.d.
11 neg n.d. 15 neg
n.d.
15 neg n.d. 26 neg
n.d.
27 neg n.d. 37 neg
n.d.
51 neg n.d. 38 neg
n.d.
52 neg n.d. 39 neg
n.d.
53 SE n.d. 40 n.d. 0% pos
55 neg 45% pos 41 neg n.d.
56 neg n.d. 42 neg
n.d.
43 n.d. 0% pos
56 n.d. 0% pos
n.d.: not done
neg: negative pos: positive
SE: Salmonella
enteritidis
Table 4: Result of
bacteriological and serological investigation flock 3
Week of live Faecal samples Serum samples
11 neg n.d.
12 neg n.d.
15 neg n.d.
21 neg n.d.
22 neg n.d.
23 neg n.d.
25 neg 0% pos
26 neg n.d.
27 neg 0% pos
40 neg 0% pos
47 n.d. 0% pos
51 SE n.d.
52 SE n.d.
n.d.: not done
neg: negative pos: positive
SE: Salmonella
enteritidis
265
Results of bacteriological and serological
investigations after introduction
of vaccination:
In year 2000 after
starting the vaccination (July 1999) SE could not be detected
in any sample
collected at the hatchery (Table 5)
Table 5: Results
of bacteriological examination in hatchery in year 2000
Samples Meconium Fluff Hatching
eggs
Environm.
swabs
Day old
poults
Total 191 93 105 71 70
Positive SE 0 0 0 0 0
In flock No. 4 in
which SE was isolated at 6th week of age. This flock was
then
vaccinated once at
26th week of age. In subsequent taken faecal samples SE
was not detected.
In all serum samples antibodies against SE were not
detected neither
after infection nor after vaccination at 26th week of age (Table
6).
Table 6: Result of
bacteriological and serological investigation of flock 4
Week of age Faecal samples Serum samples
1 neg n.d.
5 neg n.d.
6 SE n.d.
7 neg 0% pos
9 neg 0% pos
10 neg n.d.
11 neg n.d.
12 neg 0% pos
19 n.d. 0% pos
24 n.d. 0% pos
26 1. Vaccination
26 neg n.d.
36 neg n.d.
42 neg 0% pos
48 neg n.d.
51 n.d. 0% pos
52 n.d. 0% pos
n.d.: not done
neg: negative pos: positive
Flock No. 5 was
vaccinated twice at week 16 and 26. Salmonella Enteritidis was
not detected in
faecal samples through entire life. In the serological examination
2 weeks after the
first vaccination 60% of the serum samples reacted positive.
However 8 weeks
after the second vaccination no antibodies could be detected.
266
At week 44 25% and
at week 52 10% of the serum samples were serologically
positive (Table
7).
Table 7: Result of
bacteriological and serological investigation of flock 5
Week of age Faecal samples Serum samples
1 neg n.d.
2 neg n.d.
13 n.d. 0% pos
16 1. Vaccination
18 n.d. 60% pos
19 neg n.d.
26 2. Vaccination
26 neg n.d.
34 n.d. 0% pos
44 neg 25% pos
52 neg 10% pos
n.d.: not done
neg: negative pos: positive
Flocks 6, 7 and 8
were vaccinated three times at week 7, 16 and 26. In these
flocks SE could
not be detected in faecal samples through entire life. The
serological
examination showed a long lasting high antibody response. In flock
No. 6 five weeks
after first vaccination 35% of the tested serum samples
reacted positive.
After the third vaccination the serological response was nearly
100%. Flock No. 7
and 8 showed a similar good serological response (Tables 8
–10).
Table 8: Result of
bacteriological and serological investigation of flock 6
Week of live Faecal samples Serum samples
2 neg n.d.
3 neg n.d.
7 1. Vaccination
7 n.d. 0% pos
12 neg 35% pos
15 neg 50% pos
16 2. Vaccination
19 n.d. 100% pos
24 neg 60% pos
26 3. Vaccination
28 neg 0% pos
32 n.d. 95% pos
36 neg 100% pos
40 neg 100% pos
44 neg 100% pos
49 n.d. 100% pos
n.d.: not done
neg: negative pos: positive
267
Table 9: Result of
bacteriological and serological investigation of flock 7
Week of live Faecal samples Serum samples
1 neg n.d.
6 neg n.d.
7 1. Vaccination
8 neg n.d.
10 neg 0% pos
11 neg n.d.
12 neg 90% pos
16 2. Vaccination
16 neg 20% pos
25 neg 95% pos
26 3. Vaccination
27 neg n.d.
46 neg 100% pos
n.d.: not done
neg: negative pos: positive
Table 10: Result
of bacteriological and serological investigation of flock 8
Week of live Faecal samples Serum samples
1 neg n.d.
2 neg n.d.
5 neg n.d.
7 1. Vaccination
7 neg 0% pos
11 neg 20% pos
16 2. Vaccination
17 neg n.d.
26 3. Vaccination
26 neg 95% pos
49 neg n.d.
n.d.: not done
neg: negative pos: positive
Discussion
Many reports exist
about the use of live and inactivated vaccines to control
Salmonella in
poultry (Barrow and Wallis, 2000). On the other hand, only few
reports exist
concerning the use of inactivated vaccines in turkey and there are
no available
reports of the use of live vaccines in turkeys (Hafez and Jodas,
2000). McCapes et al. (1967) studied the use of a Salmonella bacterin
in turkey
breeders,
particularly to determine whether any parental resistance would be
passed to their
poults. Poults originating from S. Typhimurium vaccinated hens
exhibited
resistance to yolk-sac challenge with S. Typhimurium and S.
Schwarzengrund
(both belong to the Serogroup B), but not to S. Anatum, which
belongs to
serogroup E. According to Thain et al. (1984), administration of an
268
inactivated S.
Hadar vaccine to turkey breeding stock may be of value in limiting
the spread of this
serovar in young poults. They found that the use of this
vaccine produced
high levels of immunoglobulin G (IgG) antibodies which were
passed on through
the eggs to the poults. Also, successful vaccination of turkey
breeders for the
control of Salmonella with autogenous mineral-oil adjuvant
vaccines, prepared
from the serovars S. Sandiego and S. Arizonae, was
applied by
Nagaraja et al. (1988) Their results suggested that OMP of the
organism give
better protection than formalin-killed whole-cell bacterin.
Ghazikhanian et
al. (1984) showed that using autogenous oil-emulsion S.
Arizonae bacterin
resulted in a significant reduction of the overall egg
transmission rate
in vaccinated challenged hens compared with non-vaccinated
challenged
turkeys. The use of the subunit vaccines prepared from outermembrane
proteins (OMP)
from Salmonella Heidelberg incorporating them into
lipid-conjugated
immunostimulating complexes (ISCOMs) for protection against
homologous and
heterologous Salmonella challenge in turkeys has been
studied by Charles
et al. (1993). The re-isolation rate of Salmonella from
internal organs
after challenge with S. Heidelberg, S. Reading or S. Enteritidis
in turkeys was
completely negative for the homologous serotype and
significantly
lower for the heterologous serotype in vaccinated turkeys. The
results also
indicate that fewer turkeys shed Salmonella after vaccination with
ISCOM preparations
than after vaccination with OMP alone.
In the present
investigations Salmonella Enteritidis PT 4 was firstly isolated in
March 1999 from
routinely taken meconium samples in the hatchery. After
intensive
bacteriological investigations at the parent farm SE was isolated from
faecal samples
firstly in May 1999. SE was isolated from flock 1 and 3 during
production and in
flock 4 during brooding period. However the route of SE
introduction into
the hatchery and the parent farm could not be clarified. The
transmission and
spread of Salmonella occurs by vertical and/or horizontal
routes. Vertical
transmission can be a very important route of infection in turkey.
On the farm
Salmonella infection is transmitted horizontally frequently via faecal
contamination of
feed, water, equipment, environment and dust, in which
Salmonella can survive
for long periods. Furthermore significant reservoirs for
Salmonella are
humans, farm animals, pigeons, waterfowl and wild birds.
Rodents, pets and
insects are also potential reservoirs and transmit the
infection to birds
and between houses. In the hatchery Salmonellas can also
survive for long
periods in eggshells, meconium, dust, litter and can also be
spread by air
throughout the hatchery, resulting in rapid horizontal transmission
(Hafez and Jodas,
2000).
The vaccination
trials started in July 1999 with flock no. 4 in which SE was
isolated at week 6
and then vaccinated once at week 26. All vaccinated turkeys
did not show any
post vaccinal adverse reactions. The results of the present
investigations
revealed SE could not be detected in any examined faecal
samples from
flocks vaccinated 1, 2 or 3 times with inactivated vaccine.
Salmonellas could
also not be detected in any examined meconium and fluff
samples, one day
poults, dead in shell embryo collected in the hatchery and
originated from
the vaccinated flocks. Application of the killed vaccine
269
(Salenvac®) 3 times i.m.
resulted in good long lasting seroconversion in nearly
100% of the birds
during 54 week of life. Both humoral and cellular immunity
appear to play a
role in protection against a Salmonella infection, although the
importance of each
in the ultimate protection of the host still remains
controversial
(Barrow, 1990, Barrow et. al., 1988).
The investigations
of Tenk et al. (2000) suggest that by the use of Salenvac®
the colonization
and growth of S. enteritidis in turkey flocks can be markedly
reduced and
sometimes even prevented or avoided.
Although, in the
present field investigations no challenge trials were carried out,
it can be
conclude, that vaccination together with improvement in the production
hygiene leads to
the elimination of SE infection from the turkey breeding farm.
The vaccination
must be considered as an important support to good
management
practices and stringent biosecurity measures. Therefore the
application of
well-known and proven disease control measures are absolutely
necessary and the
effectiveness of the vaccine must be enhanced by providing
optimal management
and feeding conditions.
Additional studies
are needed to determine whether SE live vaccination in
combination with
the inactivated Salenvac® can increase the immune
response
and duration of
protection.
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