2020, Scienceline Publication

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

DOI: https://dx.doi.org/10.36380/jwpr.2020.34

Immunological Study on Salmonellae Isolated from Different

Sources

Enas A.shedeed¹, Mahmoud D. El-Hariri², Soad A.Nasef¹and J. El Jakee^2*

¹Reference Laboratory for Veterinary Quality Control on Poultry Production-Animal Health Research Institute, Dokki, Cairo.

²Faculty 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

(Sedeik et al., 2019 ).

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

birds are the main routes of transmission to humans (Prejit

et al ., 2018 ). Salmonella is categorized according to the

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):

285-291. DOI: https://dx.doi.org/10.36380/jwpr.2020.34

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

Salmonella OMPs (Prejit et al ., 2018 ).

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

Jakee et al., 2016 ).

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. Enteritidis

S. Kentucky

S.Anatum

Duckling

Poultry feed

Chicken

Group (1)

Group (2)

Group (3)

Group (4)

1,4,[5],12

1,9,12

1,2

g,m

------

Chicken

Duckling

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.,

2002 ).

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-

Wendy et al. (1998).

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.,

2019 ).

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

molecular weights of 17, 24 and 31 kDa (Maripandi and

Al-Salamah , 2010). They recorded that 14.4 and 24 kDa

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

protein (lower than 20 kDa) as recorded by Maripandi and

Al-Salamah (2010).

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

isolates (8.6%) contained only a 30.6 kDa (Maripandi and

Al-Salamah, 2010 ). More than 21 OMP bands could be

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

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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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