ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol 64 (1) 2009
SALMONELLA ENTERICA SEROVAR
ENTERITIDIS EXPERIMENTAL INFECTION IN
CHICKENS: EFFECTS OF THE INFECTIOUS
DOSE ON CROP IMMUNOGLOBULIN A
ANTIBODY RESPONSE
Ishola, O.O*. and Ogundipe, G.A.T.
Department of Veterinary Public Health and Preventive Medicine,
University of Ibadan, Ibadan, Nigeria.
Email address: olayinkaishola@yahoo.com
Phone number: 234-803-697-6193
Fax: 234-2- 8103043
* Corresponding author
ABSTRACT
In chickens the crop is an organ for storing food, and for most avian species.
Salmonella enterica serovar
Enteritidis is an important causative agent of foodborne zoonotic diseases..
This study was conducted to
determine the effects of an infectious dose of Salmonella enteritidis on the
specific immunoglobulin A (IgA)
antibody response of experimentally infected chickens. Twenty four specificpathogen-free hens were divided
into three groups of eight. A group of eight hens were orally with 1.3 x 10 8
colony forming units (cfu) of
Salmonella enteritidis and a second group received 1.3 x 104cfu of
Salmonella enteritidis per hen. The last
group was the uninfected control group.
Hens infected with 108 cfu/ml of Salmonella enteritidis produced a
significantly stronger (P<0.05) specific IgA
antibody response than those infected with 104cfu/ml and the controls. The
highest group mean optical density
values of enzyme-linked immunosorbent assay (ELISA) test for the chicken
groups infected with 108 cfu/ml
and 104 cfu/ml were 4.262 and 1.376 at weeks 4 and 3 post-infection
respectively. Levels of crop IgA antibody
were shown to be infectious dose-dependent. Hence, chickens exposed to
too few colonies of Salmonella may
not produce substantial antibodies. In order to confer optimal mucosal
immunity in chickens, they should be
exposed to an adequate vaccine dose of Salmonella enteritidis.
Keywords: Salmonella Enteritidis, immunoglobulin A, crop, mucosal
immunity, chickens, antibodies
Salmonella enterica serovar Enteritidis (Salmonella enteritidis) is an
important cause of human salmonellosis and food-poisoning associated with
consumption of contaminated chicken eggs and poultry products (1,2).
Salmonella enteritidis is one of the most frequently isolated serovars from
outbreaks of salmonellosis in poultry and humans (3,4). Poultry-associated
Salmonella food poisoning in humans is usually manifested as
gastroenteritis characterised by diarrhea, abdominal cramps and bacteremia
(5).
Infected chickens are known to produce a humoral immune response of
mainly immunoglobulin (Ig) G and IgM. Serum samples have therefore been
used widely by many authors to study IgG and IgM responses to Salmonella
infections (6-10). The predominant immunoglobulin of mucosal immunity is
IgA although IgG and/ or IgM can also be observed (11). The crop is an
enlargement or outpouching of the oesophagus proximal to the
proventriculus or glandular stomach, and functions primarily as a storage
organ (12). The crop has also been incriminated as a source of Salmonella
contamination of carcasses during processing (13). Studies have shown the
presence of IgA antibody in crops of chickens infected with Salmonella
(14,15).
However, the effects of the quantity of Salmonella that chickens are exposed
to during infection or vaccination on crop IgA levels, still need to be fully
understood. This study was therefore designed to assess the effects of an
infectious dose of Salmonella enteritidis on crop IgA antibody response by
chickens. Crop lavage technique was used to collect secretions from live
chickens over repeated periods.
MATERIALS AND METHODS
Experimental Design
Twenty-four White Leghorn 33-week old laying hens, obtained from the
specific pathogen free flock of the Southeast Poultry Research Laboratory,
United States Department of Agriculture (USDA), Athens, Georgia, USA
were divided into 3 groups of eight chickens. The birds in each group were
housed in separate rooms in a climate controlled bio-containment building.
Birds were fed antibiotic-free feed and water. They were kept off feed for 12
hours prior to sampling in order to reduce crop-bulk, thus expediting flushing
of the crop.
Experimental Infection
The hens in group 1 were challenged orally with 1.3 x 108 colony forming
units (cfu). Hens in group 2 were challenged orally with 1.3 x 104 cfu while
the remaining 8 hens were treated as the uninfected control group.
The challenge organism was a Salmonella enteritidis, nalidixic acid resistant,
phage type 13, originally of avian origin, and obtained from the National
Veterinary Service Laboratory, Ames, Iowa, USA. The organism was
maintained as frozen stocks at -20oC. Three days prior to infection, one of
the stocks was thawed and subcultured on nutrient agar (Difco) and
incubated overnight at 37oC. Single colonies were streaked onto Brilliant
Green agar containing 100μg/ml novobiocin and 10μg/ml nalidixic acid
(BGNN) and incubated overnight at 37oC.
It was then inoculated into tryptic soy broth (TSB).and incubated overnight at
37oC. The overnight culture was serially diluted in sterile normal saline
(0.85%) from 10-1 to 10-7, and plated on BGNN plates for enumeration.
Dilutions of 10-1 and 10-5 found to contain 108 cfu/ml and 104 cfu/ml and were
used to infect hens in groups 1 and 2 respectively. One ml of each dose was
given orally into the crop of each hen.
Collection of crop lavage samples
Crop lavage samples were collected using crop lavage technique (14)
involving Tygon® (Fisher Scientific) tubing and a 10ml syringe containing
5ml glycine flush (1M Tris/glycine buffer with 0.25% Tween 20, pH 7 to 8)
solution (lavage fluid). The tubing was inserted down the hen’s oesophagus
and into the crop. The lavage fluid was discharged into the crop, and then
the flush solution with the crop contents, was immediately aspirated back
into the syringe. The aspirate containing crop secretions was dispensed into
15ml sterile collection tubes and transferred on ice to the laboratory for
processing. Crop lavage samples were collected at week 0 (pre-challenge)
and at weekly intervals for 5 weeks after infecting the hens (post-challenge/
post-infection). The crop lavage samples were then centrifuged for 5 minutes
at 12,000 x g. The supernatant was harvested and frozen at -20oC until
assayed. Freezing the samples ensured prolonged viability of the
immunoglobulins present in the fluids.
Enzyme-Linked Immunosorbent Assay (ELISA)
In order to assay the crop IgA, the crop supernatant samples were thawed.
The ELISA was performed as described by Holt et al. (1999) with slight
modifications. An initial 1:2 dilution with serial twofold dilutions using
phosphate buffered saline (PBS) containing 0.05% Tween 20 (PT-buffer)
were made for each crop lavage sample. The crop lavage serial dilutions
were added to ELISA plates to which 10μg/ml Salmonella Enteritidis
lipopolysaccharide (LPS) antigen (Sigma) had been adsorbed and to which
3% polyvinyl pyrrolidone (PVP) blocker had been added to prevent
unspecific reactions. Plates were incubated at room temperature for 60
minutes after which they were washed twice using an automatic ELISA plate
washer (Bio-Tek ELX 405 microplate washer). Mouse anti-chicken IgA
(primary antibody, Southern Biotech, USA) diluted 1:1000 with PT-buffer
containing 1% bovine serum albumin (BSA) were added to plates and
incubated at room temperature for 60 minutes. Plates were then washed
after which a 1:1000 dilution of goat anti-mouse alkaline phosphatase
conjugate (secondary conjugate) in PT-buffer with 1% BSA were added and
incubated at room temperature for 60 minutes. Plates were washed again.
Lastly, the substrate, p-nitrophenyl phosphate (Sigma) at 1mg/ ml in
diethanolamine buffer was added and incubated in the dark for 30 minutes.
Absorbance was read at 405nm using an automatic ELISA reader (Bio-Tek
EL311sx autoreader).
Data analysis
Group mean optical density (OD) 405 readings of ELISA results at the 1:2
dilution for each of the infected and control groups were calculated. Titres
were determined as the last dilution that gave an OD reading 1.5 times that
of the negative control obtained previously from an uninfected chicken (14).
Significant differences (P<0.05) between the two infectious doses and the
control group and between pre-infection and the different post-infection
weeks were examined using one-way analysis of variance (ANOVA) at the
95% confidence level (16).
RESULTS
Salmonella enteritidis (SE)-lipopolysaccharide (LPS)- specific IgA antibody
response was detected at different levels for the three groups by ELISA.
Chickens challenged with 108 cfu of Salmonella enteritidis produced a
significantly stronger IgA (P<0.001) antibody response than those infected
with 104 cfu and the uninfected control hens. Significant increases in the
crop IgA response were observed for the high dose group within two weeks
of infection; the group mean OD value increased from 0.579 at pre-infection
to 3.711 by the second week to a maximum of 4.262 at week 4 post-infection
(PI) (Fig.1). While the mean OD of the 104 cfu dose group increased from
0.570 at pre-challenge (week 0) to a maximum of 1.376 at week 3 PI (Fig.
1), there was no statistical significant difference in the crop IgA antibody
response between the hens inoculated with 104 cfu and the uninfected
control hens at pre-challenge and at weeks 1, 2, 3 and 5 PI (P>0.05). At prechallenge, the antibody responses for the two dose groups and the controls
were low and did not differ significantly across the groups. A drastic
reduction in the antibody levels was observed at week 5 PI, however (Fig.1).
The mean log2 titre increased from 3.0 and 5.5 at week 1 post-infection to a
maximum of 4.0 and 7.0 at week 4 for the hens infected with 10 4 cfu and 108
cfu, respectively (Fig.2). Most of the hens challenged with 108 cfu and 104
cfu of Salmonella enteritidis gave positive titres at week 1 post-infection
which reduced to 97.5% (108 cfu) and 87.5% (104 cfu) at week 5
postinfection, and the titres of hens infected with 108 cfu (overall mean log2
titre =5.82) were significantly higher (P<0.05) than those infected with 10 4
cfu (overall mean log2 titre =3.38).
DISCUSSION
The crop immunoglobulin A (IgA) antibody response measured in this study
with the Salmonella enteritidis LPS-ELISA between chicken groups was
found to be infectious-dose dependent, and statistically significant
differences were observed in the IgA mean optical density and titre values
across the groups. The consistent production of significantly stronger IgA
antibody levels by the hens infected with 108 cfu when compared to those
infected with 104 cfu also suggests that crop IgA levels are infectious -dose
dependent, as shown in a previous study (15). An infected chicken may
produce different antibody responses depending on the number of bacteria,
the mode of colonization, and if the organism is highly invasive (8). The
significant increases observed in the IgA responses by week 2 post-infection
especially in the hens infected with 108 cfu agrees with the findings of Seo et
al. (15) and Holt et al. (17) who found strong IgA responses in Salmonella
enteritidis - infected chickens from days 12 and 10 post-infection
respectively. Also levels of anti- Salmonella enteritidis antibodies in the
crops at 3 weeks post-challenge have been shown to be significantly higher
than those from birds before infection (14). The presence of a low antibody
response in all the chicken groups at pre-infection agrees with the
observations of Seo et al. (15). Detection of crop IgA serves as a very
sensitive indicator of past exposure to S. enteritidis (18).
As the first host environment encountered by Salmonella enteritidis after
infection, the crop can therefore influence the survival and virulence of the
pathogen (1). Based on the strong IgA responses against Salmonella
enteritidis found in crops examined in this study, their proactive capacity
against Salmonella infections should not be overlooked. Another implication
of the finding of the lower antibody response in the 104 cfu compared to the
108 dose group is that during the formulation of vaccines against Salmonella
enteritidis infections in poultry, an adequate vaccine dose should be
maintained in order to stimulate an optimal antibody response by the birds.
ACKNOWLEDGEMENTS
We are grateful to the University of Ibadan management for the MacArthur
Foundation Staff Development Award given to the first author. The authors
are also grateful to Dr. P.S. Holt and Dr. L. Vaughn of the United States
Department of Agriculture (USDA), Athens, USA for their technical support
when part of this work was been carried out in Dr. Holt’s laboratory.
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