JOURNAL OF

J. Vet. Pub. Hlth., 2013, 11 (2): 83-90 VETERINARY PUBLIC

HEALTH

Development of Slide Agglutination Test for the Differentiation of Salmonella

Typhimurium Infection from Poultry Specific Salmonella Serovars
T.G. Sumithra, V.K. Chaturvedi1*, A.K. Rai, S.C. Sunita, C. Susan2 and S.J. Siju
Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, U.P.

(Received 01.12.2012; accepted 30.07.2013)

ABSTRACT
Contaminated poultry meat and eggs are the important source of Salmonella Typhimurium infection
to humans. S. Gallinarum and S. Pullorum are other frequent poultry serovars, specific to avian species,
causing infection in poultry. Thus, differentiation of these two types of infections of poultry is having a
high public health importance. In an attempt for developing a rapid and convenient differential agglutina-
tion test for this purpose, it was found that 1: 10 dilution of the prepared S. Typhimurium antigen could
differentiate this infection from poultry specific Salmonella serovars which can also be attained by undi-
luted antigen after pre-adsorption of S. Pullorum agglutinin from test sera. Since the specificity of earlier
method was limited to the first 2 min, the later was found to be better. Agglutination using the second
scheme showed a sensitivity of 85.41% with experimental infection and 100% correlation with tube
agglutination test. It was also found that 3+ or more degree of agglutination and any degree of agglutina-
tion within 5 min is equal to a STAT antibody titre of 1:30 or more. We suggest that the developed method
may be used to screen S. Typhimurium infections in poultry farms.

Keywords: Gallinarum, poultry, Pullorum, Salmonella, slide agglutination test, Typhimurium

Introduction 2009). Of these, S. Typhimurium is one of the most

Human infections caused by non-typhoidal
Salmonella have been an increasing problem in both
industrialized and developing countries for the last
several decades (Carnell et al., 2007). As
contaminated poultry meat and eggs are the most
concerned reservoirs of this infection (Cheong et al.,
2007), control measures should focus on reducing
the exposure from poultry sources. However, among
2,668 Salmonella enterica serovars (Akbarmehr et
al., 2010), only limited ones are associated with both
poultry and human salmonellosis (Paiva et al.,
*Corresponding author: vkchaturvedi@mail.com
1
Division of Biological Products, Indian Veterinary Research
Institute, Izatnagar, U.P. 243122
2
Division of Pathology, Indian Veterinary Research Institute,
Izatnagar, U.P. 243122
common serovar (Barrow and Neto, 2011). Apart
from the self limiting enteric infections of humans,
this can cause life threatening conditions in
immunocompromized patients (Dionisi et al., 2009).
S. Gallinarum and S. Pullorum are other frequent
poultry serovars but are largely specific to avian
species (Paiva et al., 2009). Thus, the differentiation
of S. Typhimurium from these serovars is essential
from public health point perspectives. But,
differentiation based on bacterial isolation is lengthy,
laborious and costly (Nagappa et al., 2007).
Concurrently, serology remains an interesting
alternative, because of its possible automation, low
cost and ability to detect chronic infections without
Salmonella excretion (Skov et al., 2002).
Slide agglutination test is a suitable serological
diagnostic technique as it is rapid, simple to perform
and inexpensive (Lilenbaum et al., 2002). However,
84 Sumithra et al.
agglutination simply based on the whole cell antigen
of S. Typhimurium can cross react with S. Pullorum/
Gallinarum due to the common 012 somatic antigen
(Poppe et al., 1992), leading to unjustified
condemnation of many poultry samples from human
consumption. So, in the present study an attempt
was made to standardize a rapid and convenient slide
agglutination test for the differential diagnosis of S.
Typhimurium infections from other poultry specific
Salmonella serovar infections. Further, the results
of the standardized test were compared with standard
tube agglutination test (STAT). Additionally we used
this test for the screening of 50 poultry received from
an organized farm in Uttar Pradesh.
Materials and Methods
Poultry
Clinically healthy chicks (4 weeks old) and adult
chicken of either sex (10-19 weeks old) were
procured from CARI, Izatnagar and maintained under
standard conditions of nutrition and management.
The absence of subclinical Salmonella infections
were confirmed serologically by using poly ‘O’ sera
(Division of biological products, IVRI) (Kauffmann,
1964).
Bacterial culture
Agar slant culture of S. Typhimurium (Culture
code E2393) obtained from National Salmonella
Centre, IVRI, Bareilly was used. This strain was
initially isolated from a 4 week old chick in Pune and
since then it is being maintained by National
Salmonella Centre. After sub culturing on Mac
Conkey agar, colonies were characterized
morphologically using Gram’s stain and serologically
with poly ‘O” sera. Molecular confirmation was done
using Salmonella specific fimA (Cohen et al. 1996)
and S. Typhimurium specific STM4497 primers (Kim
et al. 2006). After characterization glycerol stock was
made and stored at -20oC.
Production of S. Typhimurium colored antigen
Hundred millimeter of nutrient broth was
inoculated from the master seed and incubated at
37oC for 24 h. After testing for purity, 3 ml of the
culture was used for sowing Roux flasks each
containing 125 to 150 ml of nutrient agar. Following
incubation at 37oC for 72 h, the growth was harvested
with 0.5% formal saline and examined for purity.
Subsequent to passing through muslin cloth sieve,
the filtrate was kept at 37oC for 24 h for complete
inactivation and then tested for sterility. The harvest
was then centrifuged at 4000 rpm for 30 min in a
cooling centrifuge. One ml of the packed cell was
then re-suspended in 10 ml of resuspending solution
(1% formalin, 1% KH 2 PO4 and 0.85% sodium
chloride) and mixed thoroughly. To 100 ml of this
antigenic suspension 3 ml of 1% aqueous solution
of crystal violet was added. After mixing, the colored
antigen was allowed to stand 48 h and then stored
in amber colored bottle at 4oC.
Production of S. Typhimurium positive serum
An aluminium hydroxide gel vaccine of S.
Typhimurium was prepared and used for raising S.
Typhimurium positive serum in chicken. For this,
O.D. of the above bacterial harvest was adjusted to
Brown opacity tube no. 9 with sterile normal saline
followed by the adjustment of pH to 6.2-6.4 by
sterilized sodium hydroxide solution. Forty parts of
autoclaved 3% aluminium hydroxide gel was then
added to 60 parts of the bacterial suspension and
agitated slowly at room temperature for 4 h. After
testing safety in 3 chicks, 0.5 ml was injected i/m as
the first dose of antigen in adult chicken. Thereafter
a booster dose was given at 14th day. Seven days
after second injection serum was collected and stored
at -70oC.
Standardization of agglutination test
The cross reaction between S. Typhimurium and
S. Pullorum was confirmed by observing the
agglutination between Typhimurium antigen and
Pullorum specific serum (Division of biological
products, IVRI). The colored antigens of S.
Typhimurium (prepared as above) and S. Pullorum
(Division of biological products, IVRI) were
resuspended as 5% bacterial cell concentration and
used for standardization by two schemes. In the first
scheme, undiluted and each tenfold dilutions of S.
Typhimurium antigen were used for agglutination
against control, known positive S. Typhimurium and
S. Pullorum serum. The highest dilution showing
 Development of slide agglutination test for the differentiation of Salmonella
definite positive and negative pattern with S.
Typhimurium and S. Pullorum serum, respectively,
was found out. In the second scheme, an equal
amount of S. Typhimurium positive sera and S.
Pullorum colored antigen was mixed together and
incubated at 37oC for 1 h to allow the agglutination
reaction between the shared antigens and antibodies.
The agglutinated mass was removed by
centrifugation at 10000 rpm for 5 min in a cooling
centrifuge. Thirty µl of the resultant sera were used
for agglutination against of S. Typhimurium and
Pullorum antigens. In both methods, the degree of
agglutination were recorded as 4+ (one solid
aggregate or clump of cells), 3+ (several large
aggregates with clear background), 2+ (small to
medium sized aggregates and clear background) 1+
(small aggregates, turbid bluish background).
Experimental infection
For determining the sensitivity of this test in
comparison with STAT, birds were experimentally
made positive to S. Typhimurium infection. For this,
a total of 18 birds were equally divided into three
groups. Group A were inoculated with 0.5 ml of an
inactivated aluminium hydroxide vaccine, prepared
as described earlier. Group B were orally inoculated
with a single dose 1 ml of 18 h old broth culture
(1.47x 109 CFU/ml) of S. Typhimurium. Group C were
kept as control. Each group was housed in a separate
room. Serum was collected before inoculation and
at weekly intervals for 4 week post-inoculation. The
standardized agglutination test was performed on
all sera samples. Sensitivity of the test was calculated
as the number of test positive hens divided by the
total number of hens infected with S. Typhimurium
(Jouy et al., 2006).
Comparison with STAT
The whole cell antigen prepared by re-
suspending the formalized S. Typhimurium cell pellet
in normal saline solution to give turbidity equivalent
to Brown’s opacity tube No.2 was used as antigen
for STAT. The protocol of Barsoum and Awad (1972)
was followed with minor modifications. Briefly, after
removal of the S. Pullorum agglutinin from test sera
after adsorption, serial two fold dilutions of serum
samples were made in normal saline. To each serum
85
dilution, 0.5 ml of the antigen was added and racks
containing the tubes were shaken and incubated in
water bath at 37oC for 18 h. The tubes were then
examined for agglutination in front of a light source.
STAT titre was expressed as the reciprocal of highest
dilution showing 100% clearing.
Statistical analysis of the results was done using
SPSS 17 software to find out the mean values of
STAT titre and correlation between STAT and the
proposed agglutination test.
Validation with field samples
As a field trial, 50 poultry serum samples
collected from birds maintained in an organized farm,
Izatnagar were screened by the standardized slide
agglutination test.
Results and Discussion
S. Typhimurium, a zoonotic enteric pathogen of
worldwide importance, has the ability to infect a wide
range of hosts (Carnell et al., 2007). In humans, the
infection is often acquired through consumption of
infected poultry meat or eggs (Beal et al., 2004) and
infection can range in severity from subclinical
infection, through mild diarrhoea, to severe systemic
disease (Carnell et al., 2007). Conversely, the
infection in poultry more than 3 days of age, results
in a persistent enteric infection without clinical
disease (Beal et al., 2004). Consequently, the
infected flocks are difficult to identify and represents
a major source for entry into the human food chain
(Beal et al., 2004). Hence, detection of S.
Typhimurium infected poultry flocks is necessary to
control the infection among human population
(Nagappa et al., 2007). Although sensitivity of
bacteriological methods is generally sufficient, it
cannot recognize intermittent excreters (Bager and
Petersen, 1991). At the same time, due to the high
concentrations of circulating IgG persisting for
several months following Salmonella infection,
serological tests can detect such intermittent
excreters (Barrow, 1992). The commonly used
serological tests for Salmonella include rapid serum
agglutination test (RSAT), tube agglutination,
microagglutination, and enzyme-linked
immunosorbent assay (ELISA) etc (Barrow, 1992;
86 Sumithra et al.

Table 1. Standardization of agglutination with various dilutions of S. Typhimurium antigen

S. Typhimurium serum S. Pullorum serum* Control serum

S. Typhimurium Undiluted ++++ ++ -

antigen 1 : 5 dilution ++++ + -
(5 % suspension) 1: 10 dilution ++++ - -
1: 20 dilution +++ - -
* The results within 2 min are given

Table 2. Results of rapid plate agglutination test in experimental S. Typhimurium infection

Group Identification code Days post infection
of poultry 7 14 21 28
A 1401 +++ (4-5 min) +++ (3-4 min) ++ (6-7 min) -
1402 - +++ (3-4 min) ++++ (1 min) +++ (3-4 min)
1405 +++ (5 min) ++++ (4 min) +++ (3-4 min) ++ (5-6 min)
1412 +++ (3-4 min) +++ (3-4 min) +++ (3-4 min) -
1414 +++ (3-4 min) +++ (3-4 min) ++ (6-7 min) -
1423 ++ (7 min) +++ (3-4 min) +++ (1 min) ++ (6-7 min)
B 1392 ++++ (1 min) ++++ (1 min) +++ (3-4 min) +++ (3-4 min)
1394 - +++ (3-4 min) +++ (1 min) ++++ (1 min)
1372 +++ (4-5 min) ++++ (3 min) +++ (3-4 min) +++ (4-5 min)
1375 ++ (3-4 min) +++ (3-4 min) +++ (3-4 min) ++++ (2 min)
1393 +++ (3-4 min) ++++ (1 min) ++++ (1 min) +++ (2-3 min)
1382 +++ (3 min) ++++ (1 min) +++ (1 min) +++ (3-4 min)

Table 3. Results of STAT in experimental S. Typhimurium infection

Group Identification code Days post infection
of poultry 7 14 21 28

A 1401 1.477 1.6020 1.301 0.000

1402 1.000 1.477 1.7781 1.6020
1405 1.6020 1.6989 1.477 1.000
1412 1.477 1.6020 1.6020 0.000
1414 1.6020 1.6020 1.301 1.000
1423 1.000 1.6020 1.6989 1.301
Mean±SE 1.36±0.12b 1.60±0.03b 1.53±0.08b 0.82±0.27a
B 1392 1.7781 1.7781 1.6989 1.6989
1394 0.000 1.477 1.6989 1.7781
1372 1.477 1.6989 1. 6020 1.427
1375 1.00 1.477 1.6020 1.7781
1393 1.6020 1.7781 1.7781 1.6020
1382 1.477 1.6989 1.477 1.301
Mean± SE 1.22±0.26a 1.65±0.06a 1.64±0.04a 1.60±0.08a
 Development of slide agglutination test for the differentiation of Salmonella
Table 4. Percentage of birds positive for Salmonella Typhimurium antibodies*
Group Plate agglutination test
A 83.33%
B 87.5%
C 0
* Six hens per group each tested for 4 weeks post-inoculation, thus a total 24 samples per group.
Fig. 1. PCR amplification of Salmonella
Fig. 2. PCR amplification of S. Typhimurium specific
fimA primer specific STM4497 primer
87
STAT
91.67%
95.83%
0
Fig. 3. Rapid slide agglutination test after pre-
adsorption of S. Pullroum agglutinin. A:
Agglutination with S. Typhimurium serum B:
Agglutination with S. Pullorum serum
Poppe et al., 1992). Of these, RSAT carry advantages
over the other tests such as ease of performance
and requirement of less time, space, antigen, serum
etc (Barsoum and Awad, 1972; Nagoba and Nagoba,
2008).
S. Gallinarum and S. Pullorum are two other
frequent poultry serovars, neither of which can infect
humans thus have no public health significance
(Pomeroy et al., 1991). At the same time due to the
similar antigenic structure of these two serovars
serological tests based on S. Pullorum antigen are
used to detect birds infected with both serotypes
(Oliveira et al., 2004). The most of routine serological
tests for S. Typhimurium (1, 4, 5, 12; i; 1, 2) infection
are based on whole cell antigen of this bacteria which
can make cross reactions with S. Pullorum/
Gallinarum (9, 12; - ; -) due to the common 012
somatic antigen (Poppe et al., 1992; Barrow, 1992;
Barrow et al., 1992). Cross reaction between these
two infections can lead to false positive information
about S. Typhimurium infection (Spickler et al.,
88 Sumithra et al.
2010). Thus ultimately result in unjustified
condemnation of many poultry samples from human
consumption. Keeping these facts in view an attempt
was made to develop a rapid and convenient slide
agglutination test for the differential diagnosis of S.
Typhimurium infections from poultry specific
Salmonella infections.
During the characterization of the procured
bacterial culture typical cultural, morphological,
molecular (Fig. 1 and 2) and serological
characteristics of S. Typhimurium were observed.
The harvest produced from the culture of these
bacteria was pure and did not yield any growth on
nutrient agar denoting their sterility. Then cross
reaction between S. Typhimurium and S. Pullorum
was confirmed by observing the agglutination
between Typhimurium antigen and Pullorum specific
serum. There was 2+ degree of agglutination within
1-2 min indicating the need for a differential test.
During optimization of agglutination, it was found
that both 1: 10 and 1: 20 dilution of 5% S.
Typhimurium antigen could produce definite positive
pattern with S. Typhimurium serum and negative
pattern with S. Pullorum serum within 2 min (Table
1). However, the specificity with S. Typhimurium
serum was limited to the first 2 min only. This lack
of specificity was not unexpected since, it is already
established that during agglutination tests for
diagnosis of any infection the stained antigen may
react with the non-specific antibody present in the
serum with the lapse of time (Arefin et al., 2011).
Moreover, 012, the dominant somatic antigen of S.
Typhimurium is also carried by many of the most
prevalent Salmonella serovars in layer flocks (Poppe
et al., 1992). There comes the advantage of our
second optimized method of agglutination in which
it was found that the sera after the removal of the
shared antigens could give definite positive pattern
with S. Typhimurium antigen and negative pattern
with S. Pullorum antigen with maintenance of
specificity after 2 min also (Fig. 3). Thus, we
recommend the use of 1: 10 dilution of the prepared
S. Typhimurium antigen or undiluted antigen after
pre-adsorption of S. Pullorum agglutinin from test
sera for serological screening of S. Typhimurium
infection in poultry with more priority for the second
method.
As the definitive conclusion regarding S.
Typhimurium infection can be well attained by using
undiluted antigen after pre-adsorption of S. Pullorum
agglutinin from test sera, this test was used for
further studies. In the attempt for determining the
sensitivity of this test, the results obtained are given
in the Table 2-4. The overall sensitivity was found to
be 85.415%.
Thus, in experimental infection/ immunization
studies, almost similar results were obtained in both
STAT and new RSAT although the frequency of
positive results was consistently greater with tube
tests than with plate tests. Some prior investigations
likewise showed that serum tube tests can detect S.
Pullorum infected chickens more frequently than
plate tests (Gast, 1997). However, comparative
conclusions about STAT and RSAT should also take
into account that RSAT test is easy and cheap to
perform with limited use of space, antigen and
serum. Moreover, RSAT results showed 100%
correlation with STAT results (correlation was
significant at 0.01 level). This is in accordance with
the previous studies by Faydi and Al Khalil, (1992)
on brucellosis and Barsom and Awad, (1971) on
typhoid infection of human who found that plate
agglutination correlated well with tube agglutination
results. In the statistical analysis there was 100%
correlation between the agglutination test and STAT
and the correlation was found to be significant at
0.01 levels. In the attempt for correlating the degree
of agglutination with STAT titre, it was found that an
agglutination of +++ or more require a STAT titre of
1:30 or more. Since there is much of the potential
for human error with subjective interpretation of
degree of standard agglutination test, an attempt was
made for correlating the time of appearance of
agglutination and STAT titre. It was found that a STAT
titre of 1:30 or more is required for the appearance
of clumps within 5 min in the standardized plate
agglutination method. During the validation of the
RSAT with field samples 2 samples out of 50 samples
(4%) were found positive. The sero-prevalence
although having only 4%, it is a matter of concern
due to the public health significance of this organism.
However, various other tests for detecting S.
Typhimurium antibodies (Barrow, 1992; Poppe et
al., 1992; Kles et al., 1993) have been reported to be
 Development of slide agglutination test for the differentiation of Salmonella
more sensitive than RSAT and STAT. So we also
recommend that the principle, pre-adsorption of S.
Pullorum agglutinin from test sera has to be applied
before the execution of these tests to avoid
complication in the interpretation of results.
A crystal violet stained S. Typhimurium antigen
from a local isolate was successfully developed and
standardized by two methods for the differential
diagnosis of zoonotic S. Typhimurium infection from
poultry specific Salmonella serovars. Along with this,
the present study also point out the importance of
pre-adsorption of nonspecific agglutinin from test
sera or dilution of antigen and interpretation within
2 min during serological screening of S.
Typhimurium infections in poultry farms.
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