JWPR  
Poultry Research  
J. World Poult. Res. 10(2S): 285-291, June 14, 2020  
Journal of World’s  
Research Paper, PII: S2322455X2000034-10  
License: CC BY 4.0  
Immunological Study on Salmonellae Isolated from Different  
Sources  
Enas A.shedeed1, Mahmoud D. El-Hariri2, Soad A.Nasef1 and J. El Jakee2*  
1 Reference Laboratory for Veterinary Quality Control on Poultry Production-Animal Health Research Institute, Dokki, Cairo.  
2 Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.  
*Corresponding author’s Email: jeljakee@yahoo.com; ORCID: 0000-0002-5299-3783  
Received: 19 Feb. 2020  
Accepted: 25 Mar. 2020  
ABSTRACT  
Salmonella infection is a critical veterinary and medical problem worldwide and is a major issue in the food industry.  
Non-typhoidal Salmonella is known as an important pathogen causing gastroenteritis. The Outer Membrane Proteins  
(OMPs) of Gram negative bacteria are significant for virulence, host immune responses and drug therapy targets.  
Enhanced diagnosis of live poultry colonized with Salmonella species is required to avoid foodborne diseases. The  
present study was based on molecular characterization of OMPs among four Salmonella serovars (S. Typhimurium,  
S. Enteritidis, S. Kentucky and S. Anatum) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The  
OMPs profiling showed more than 70 protein bands ranged in size from 208 kDa to below 16 kDa which were  
detected using Total Lab 1D 12.2 software. All Salmonella strains had a band at 54-60 kDa, 45-53 kDa, 36-39 kDa  
and 26-31 kDa. Eleven strains exhibited a band at 41-46 kDa and 33-35 kDa. Nine strains had a band at 61-69 kDa.  
Eight strains exhibited a band at 135-145 kDa and 72-79 kDa. Seven strains had a band at 108-123 kDa and 83-91  
kDa. In the Western blot analysis, the prepared hyperimmune anti serum of each Salmonella serovars reacted with the  
35 kDa protein band. It is concluded that the identification of novel immunogenic proteins would be useful in  
developing ELISA-based diagnostic assays with a higher specificity.  
Key words: Outer Membrane Proteins, Salmonella, SDS-PAGE, Western blotting.  
INTRODUCTION  
antigen. The use of a number of antisera directed to some  
of those surface antigens of Salmonella constitutes a  
universal subtyping method called serotyping (Quintana-  
Ospina et al., 2018). The predominant serotypes present in  
Egyptian poultry farms are S. enterica serovar  
Typhimurium and S. enterica serovar Enteritidis  
Non-typhoidal Salmonella is a fundamental cause of  
food-borne disease globally. It is a universal public health  
interest, reporting more than 94 million cases and 115,000  
deaths every year, with disproportionate influence in  
developing countries. Salmonellae were revealed in 5% of  
minced meat samples, 10% of the 20 burger samples, 35%  
of sausage samples and 25% of poultry products.  
Salmonella isolates were revealed as S. Infantis, S. Lagos,  
S. Bolombo, S. Cerro, S. Enteritidis, S. Kentucky, S.  
Newlands, S. Newport, S. Saintpaul, S. Sandiego, S.  
Senftenberg and S. Typhimurium (EI Jakee et al., 2014).  
Another serious health problem that affects  
antimicrobial treatment is the existence of multidrug-  
resistant (MDR). Many studies show that infections  
Salmonellosis is the most commonly reported foodborne  
zoonotic disease in humans that can cause chronic illness,  
mortality and societal expenses. The causative agent of  
salmonellosis includes a variety of Salmonella enterica  
serovars. While more than 2500 serovars of S. enterica  
have been reported, among those S. Typhimurium was  
reported to be second most prevalent serovar of zoonotic  
significance isolated from humans worldwide. Several  
countries are confronting this public health crisis due to its  
resistance to antimicrobial agents and rapid transmission  
of Salmonella via food and water. These organisms are  
associated with poultry gut, thus the consumption of  
contaminated poultry meat, egg and contact with infected  
different antigens found in the cell wall of bacteria, the O  
antigen is recognised as somatic antigens and the H  
antigen is constituted by polymerized subunits of flagellin,  
while the virulence-associated antigen expressed in the  
surface of some Salmonella strains is known as Vi or K  
To cite this paper: Shedeed EA, El-Hariri MD, Nasef SA and El Jakee J (2020). Immunological Study on Salmonellae Isolated from Different Sources. J. World Poult. Res., 10 (2S):  
285  
Shedeed et al., 2020  
produced by MDR strains are more serious than these  
bacteria surface and has special importance as among the  
potential protective immunity targets. Recent researches  
have tended to focus on the Outer Membrane Proteins  
(OMPs) proposing the presence of Salmonella protective  
immunogenic elements. The OMPs have been identified to  
be immunogens for evolving active/protective immunity  
against Salmonella and thus, have tremendous possibility  
to be used in vaccination. OMPs have been inspected as  
potential candidates for vaccine, virulence factors and  
those surface exposed proteins play a vital role in  
pathogenic mechanisms including host cells motility,  
adhesion and colonization, injection of toxins and cellular  
proteases, and the formulation of channels for the  
sweeping of antibiotics (Singh et al., 2017).  
The OMPs are effective immunogens on the  
bacterial surface, which have been used in many trials to  
check their ability as a vaccine candidate in poultry.  
Studies have been focused on evolution of OMPs  
diagnostic antigen. OmpC, OmpF, OmpD are the principal  
produced by susceptible strains (Djeghout et al., 2017).  
Food-borne salmonellosis is a massive public health  
problem not only in the developing countries but also in  
industrialized countries, resulting in increasing incidence  
of enteric diseases, hospitalizations and even deaths every  
year globally. As one of the most common food borne  
pathogens, Salmonella infects more than 160,000  
individuals in the European Union annually, with a  
morbidity rate of 35 cases per 100,000.6 Salmonella was  
the second etiologic agent which is laboratory confirmed  
responsible for 229 (30%) recorded outbreaks of food  
poisoning in the United States and the economic cost of  
Salmonella infections is $2.4 billion annually (Wang et al.,  
2017). Several diagnostic tests for detecting of Salmonella  
infections in poultry were developed. S. Typhimurium  
(0.6) and S. Enteritidis (0.5%). were isolated from eggs (El  
Cultural isolation is the standard technique for  
detecting salmonellae in hatcheries and breeding flocks.  
Cultural procedures for the detection of Salmonella,  
however, are laborious, costly, time-consuming and  
individual birds intermittently excrete S. enterica or may  
remove the infection completely. Therefore, the designing  
of dependable screening tests would help identify  
Salmonella presence in hatchery environments and flocks.  
Serological approaches such as ELISA could help to  
identify the existence of infected and carrier birds and also  
silent transmission throughout the flock, that can be  
missed by traditional bacteriological methods because of  
the sporadic Salmonella shedding (Manoj et al., 2015).  
Improvement of detection methods and development  
of new vaccines would simplify the detection,  
characterization, and validation of previously unbeknown  
immunogenic proteins (Meyer et al., 2012). The outer  
membrane is a continued structure on Gram-negative  
The present study aimed to characterize the OMPs of  
Salmonella serovars (S. Typhimurium, S. Enteritidis, S.  
Kentucky and S. Anatum) collected from different sources  
and to identify antigenic proteins by Western blotting.  
MATERIALS AND METHODS  
Ethical approval  
The study was approved by the Institutional Animal  
Care and Use Committee of Cairo University, Giza, Egypt  
(Vet CU20022020145).  
Bacterial strains  
Twelve Salmonella isolates collected from duckling,  
chicken, and poultry feed were obtained from the reference  
laboratory for veterinary quality and control on poultry  
production (Table 1).  
Table1. Salmonella strains used in the present study  
Antigenic structure  
Source of  
Groups  
Serotype  
Flagellar (H) antigen  
Somatic  
(O) antigen  
strains  
Phase1  
Phase2  
S. Typhimurium  
S. Typhimurium  
S. Typhimurium  
S. Enteritidis  
S. Enteritidis  
S. Enteritidis  
S. Kentucky  
S. Kentucky  
S. Kentucky  
S.Anatum  
Duckling  
Duckling  
Poultry feed  
Chicken  
Group (1)  
Group (2)  
Group (3)  
Group (4)  
1,4,[5],12  
1,9,12  
i
1,2  
g,m  
i
------  
z6  
Chicken  
Duckling  
Duckling  
Duckling  
Chicken  
Chicken  
Chicken  
8,20  
S.Anatum  
3,{10}{15}{15,34}  
e,h  
1,6 [z64]  
S.Anatum  
Chicken  
286  
J. World Poult. Res., 10(2S): 285-291, 2020  
Laemmli (1970). The OMP extracts were solubilized in  
Confirmation of the isolates  
The collected isolates were tested for purity using  
xylose lysine deoxycholate (Oxoid). Confirmation of the  
isolates using biochemical characterization and serological  
identifications (with agglutination tests with specific O  
and H antisera, and classified according to the Kauffmann-  
White-Le Minora scheme) were performed (Quinn et al.,  
treatment buffer containing β-Mercaptoethanol. Samples  
boiled in a water bath for 90 seconds then quickly  
transferred to ice water. The separation was carried out at  
a constant current 150V per gel for about 4 hrs. Gels  
were stained with Coomassie Brilliant blue R-250  
staining solution for 4 hrs at room temperature. After  
staining, the slab gel was immersed in destaining solution  
repeatedly until the background became clear (about 3  
hours). Finally, gel was washed with distilled water. The  
gel was viewed and photographed under gel  
documentation. The pictures of gel and marker were  
loaded on computer program (TollLab) to calculate the  
molecular weights of peptide bands.  
Real-time PCR  
Molecular confirmation of Salmonella isolates was  
done with Salmonella specific primers targeting the invA  
gene by real-time PCR. DNA Extraction performed  
according to the QIAamp DNA mini kit. Specific primers  
were used and the cycling program was done according to  
Daum et al. (2002). Master Mix was prepared according to  
the Quantitect probe Real-time PCR kit. Results were  
monitored by the Stratagene MX3005P set.  
Preparation of hyperimmune sera against  
Salmonella serovars  
Hyperimmune antiserum was obtained from 20  
chicks (5 chicks for each strain) inoculated IP with 1011  
formalin killed Salmonella serovars (S. Typhimurium, S.  
Enteritidis, S. Kentucky and S. Anatum) solubilized in  
Auspharm adjuvant as emulsion (0.5 mg/dose) at 19 and  
33 days of age, and an oral booster at 47 days of age.  
Blood was collected after 7 days and serum was prepared  
and stored at 200C according to the protocol of Muir-  
Isolation of outer membrane proteins  
The OMPs from Salmonella were isolated as  
described by Verdugo-Rodriguez et al. (1993) with some  
modifications. Twenty Four hours cultures of bacterial  
cells in nutrient broth were centrifuged at 1,400xg at 4°C  
for 10 minutes. The bacterial cell pellet thereafter,  
resuspended in phosphate-buffered saline (PBS, pH 7.4),  
and sonicated at a setting of 20kHz or 20,000 cycles/sec  
(Vi bra Cell sonicator, Sonic & Material Co., Danbury,  
Connecticut, USA). Sonicated cells were centrifuged at  
1,400 x g at 4°C for 10 minutes, and centrifuge the gained  
supernatant at 100,000 x g at 4°C for 30 minutes and the  
pellet resuspended in 20 ml of PBS, pH 7.4 containing  
20% Triton X-l00 and incubated at 37°C for 20 minutes.  
The centrifugation step was repeated and the pellet was  
resuspended in 1 ml of PBS, pH 7.4, and stored at -20°C  
until use. The protein content was analyzed by the  
NanoDrop®ND-1000 Spectrophotometer (NanoDrop  
Technologies, Wilmington, DE USA) at 280 nm at  
reference laboratory for veterinary quality and control on  
poultry production. It was suitable for performing  
Electrophoresis.  
Western blot  
Proteins from culture supernatant gels were blotted  
on nitrocellulose membranes in 25 mM Tris-HC1, 192  
mM glycine buffer, pH 8.3, containing methanol 20% v/v  
(Neal, 1981). The transfer was influenced by a current of  
100 mA overnight in a Bio-Rad Trans blot cell. Free  
protein sites were saturated by incubation in blocking  
buffer containing newborn calf serum (Gibco) 10% v/v in  
phosphate-buffered saline, pH 7-4, Triton X-100 0.2%  
v/v for 30 min. The nitrocellulose membrane was then  
incubated in anti-Salmonella diluted 1: 20000 PBS1 in 50  
in blocking buffer for 1.5 h. After washing three times for  
15 minutes each in phosphate-buffered saline, pH 7.4,  
Triton X-100 0.2% v/v the nitrocellulose membrane was  
incubated with Rabbit -anti chicken horse reddish  
peroxidase-conjugated Ab (KPL) 1:5000 (Secondary Ab)  
The paper was washed afterward and a chromogen  
substrate containing tetramethylbenzidine was added.  
Salmonella species OMP separation by sodium  
dodecyl sulfate-polyacrylamide gel electrophoresis  
Analysis of protein profiles of the Salmonella  
serovars (S. Typhimurium, S. Enteritidis, S. Kentucky,  
and S. Anatum) was done by sodium dodecyl sulphate  
polyacrylamide gel electrophoresis which was performed  
on 12% separating and 5% stacking gels using a  
discontinuous buffer system in a biorad Protein II vertical  
unit (BioRad, Richmond, CA, USA) as defined by  
Calculation of molecular weights of the proteins  
The relative migration values of the migrated  
protein fraction were calculated in relation to protein  
marker by Total Lab 1D 12.2 software.  
287  
Shedeed et al., 2020  
chickens and turkeys were compared, no differences were  
RESULT AND DISCUSSION  
found among the isolates within this serovar (Aksakal,  
2010). S. Enteritidis with different OMPs bands were  
exhibited with a molecular weight ranged from 5-90 kDa  
and the major OMPs profiles of all S. Enteritidis isolates  
were homogenous with different expression in intensity of  
protein was observed by Maripandi and Al-Salamah  
(2010). The whole cell proteins of S. Typhimurium and S.  
Enteritidis showed similarity in analysis by SDS PAGE  
analysis, both strains yielded major bands at 71.4, 67.7,  
44.0, and 30.3 kDa (Aksakal, 2010).  
In the present study, all S. Anatum strains had a band  
62-56, 54-52, 46-42, 41-40, 38-37, 35-33, 29-26, and 18-  
16 kDa. And all S. Kentucky strains had a band at 62-60,  
54-51, 44-42, 38-37and 29-28 kDa. Salmonella Kentucky  
is among the most frequently isolated S. enterica serovars  
from food animals in the United States (Haley et al.,  
Infection with Salmonella is a significant medical  
and veterinary problem globally causing major concern in  
the food industry. This study implemented the Western  
blot technique to detect the presence of antigenic proteins  
of Salmonella strains. The result showed that  
hyperimmune antiserum of each Salmonella serovar  
reacted with the OMP 35 kDa protein band (Figure 3).  
Another study carried out Western blot analysis against  
OMP of S. Enteritidis, serum antibodies from chicken  
infected with S. Enteritidis reacted with protein band at  
molecular weight 14.4 and 24 kDa, while antibodies raised  
against S. Typhimurium reacted with protein bands at  
proteins were immune response protein and can use for  
vaccine development.  
Salmonellae are significant gastrointestinal pathogens that  
pose a global threat to public health. A total of 12  
Salmonella strains were included in the study, 5 of them  
were isolated from duckling, 6 from chicken and one from  
poultry feed. The isolates were confirmed to be  
salmonellae using conventional and molecular methods  
(Figure 1). The outer membrane is a persistent feature on  
Gram-negative bacteria surface and has particular  
significance as one of the potential targets for protective  
immunity. Determination of the protein content plays an  
important role in bacterial classification, identification,  
typing, and comparative studies. New searches on OMPs  
have suggested the presence of Salmonella protective  
immunogenic components (Singh et al., 2017).  
The results of SDS-PAGE showed that more than 70  
protein bands ranged in size from 208 kDa to below 16  
kDa. (Figure 2). All Salmonella strains had a band at 54-  
60 kDa, 45-53 kDa, 36-39 kDa, and 26-31 kDa. Eleven  
strains had a band at 41-46 kDa and 33-35 kDa. Nine  
strains had a band at 61-69 kDa. Eight strains had a band  
at 135-145 kDa and 72-79 kDa. Seven strains had a band  
at 108-123 kDa and 83-91 kDa.  
Protein bands of 78.1, 51.2, 41.5, 37.3, 35.1, 33.9,  
30.7, 27.6, 25.4, and 24 kDa were detected in all  
Salmonella serovars and protein bands of 78.1, 51.2, and  
41.5 kDa appeared as major bands in all strains (Aksakal,  
2010). The intense protein region which occupied the  
range from14 and 45 kDa constituted the Salmonella  
specific OMP bands, the higher molecular weight region  
(higher than 45 kDa) and at the lower molecular weight  
region (lower than 14 kDa) were bands related or  
associated to the OMP or residues of flagella and pilus  
All S. Typhimurium had a band at 96-84, 75-72, 69-  
63, 47-45, 43-42, 37-36, 34-33, 27-26 and 22-20 kDa. The  
majority of S. Typhimurium isolates (74.3%) contained  
two OMPs of 30.6 and 34.6 kDa, 6 isolates (17.1%)  
carried three OMPs of 27.2, 30.6 and 34.6 kDa and three  
resolved from Salmonella Typhimurium, Salmonella  
Breanderp and Salmonella Lomita ranging in size from  
61.0 kDa to 7.5 kDa (Osman and Marouf, 2014).  
The finding of the present study highlighted that 35  
kDa OMP of Salmonella serovars (S. Typhimurium, S.  
Enteritidis, S. Kentucky and S. Anatum) is an immune-  
response protein. This protein can be used for vaccine  
preparation in future.  
Jaradat and Zawistowski (1998) demonstrated the 35  
kDa OMP contained an antigen common for all tested  
Salmonella species except atypical species such as S.  
Arizona. The results of the protection studies conducted by  
El-Tayeb et al. (2019) indicated that the highest protection  
was observed using the 38 kDa OMP, which provided  
100% protection to mice challenged with 50× LD50 of  
Salmonella Typhimurium SA3 and 75% protection to mice  
subjected to an even higher bacterial challenge of 100×  
LD50. Therefore, 38 kDa OMP is a promising candidate  
for the vaccine development against S. Typhimurium.  
Pandey et al. (2018) concluded that OMP 28 may be  
proven to be an effective candidate for the development of  
Among S. Enteritidis all strains had a band at 91-82,  
72-67, 59-55, 45-43, 39-38, 37-35, 25-29, and 16 kDa.  
When the protein profiles of S. Enteritidis originating from  
288  
J. World Poult. Res., 10(2S): 285-291, 2020  
recombinant DNA vaccines against salmonellosis.  
suitable screening tool for serological monitoring of  
poultry flocks. Recently, Li et al. (2019) established  
indirect ELISA using the IpaJ protein (a new antigen  
reported to be specific to S. Pullorum, and not detected in  
S. Gallinarum and S. Enteritidis) is a novel method for  
specific detection of S. Pullorum infection, and contribute  
to eradication of Pullorum disease in the poultry industry.  
Antigenic bands of Salmonella spp. of 10, 15, 17 and 40  
kDa and 10, 17, 25, 37 and 75 kDa were detected in 15 out  
of 18 (83.3 %) and 4 out of 18 (22.2 %) samples from  
chicken carcasses and egg surface, respectively. Quintana-  
Ospina et al. (2018) suggested that rOmpC evident by a  
single protein band of 43 kDa based indirect ELISA as a  
Figure 1.Amplification curves of real time PCR for detection of invA gene of studied Salmonella serovars by Stratagene  
MX3005P.  
Figure 2. Sodium dodecyl sulphate poly acrylamide gel electrophoresis of outer membrane proteins extracted from different  
Salmonella strains and stained with Coomassie Brilliant Blue R-250. A): Lanes 1, 2 and 3: S. Typhimurium, Lanes 4 and 5: S.  
Enteritidis and Lane M: Molecular weight standards. B) Lane 6: S. Enteritidis, Lanes 7, 8 and 9: S. Kentucky, Lanes 10, 11  
and 12: S. Anatum and Lane M; Molecular weight standards.  
289  
Shedeed et al., 2020  
Author’s Contributions  
All authors contributed equally to this work  
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DECLARATIONS  
Acknowledgments  
This work was supported by Dr. Jakeen Eljakee, Dr.  
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