Isolation of O & H Antigen from Salmonella typhi

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Aim: Isolation of O & H Antigen from Salmonella typhi

BACKGROUND

Typhoid fever, paratyphoid fever, and other food-borne illnesses are all brought on by the Salmonella genus of rod-shaped, Gram-negative, non-lactose fermenting Enterobacteriaceae. Subspecies enterica serotypes Paratyphi A, B, and C are responsible for paratyphoid fever.

The Widal test is the most popular diagnostic technique for determining salmonella infection. The Widal test finds out whether a person’s blood contains antibodies against Salmonella enterica antigens.

Salmonella has 2 types of antigens, i.e., flagellar antigen is known as H antigen and somatic or lipopolysaccharide antigen is known as O antigen. research on Salmonella Enteritidis recently have shown that the lipopolysaccharide (LPS) is a virulence factor and that a certain LPS molecule structure may be an accurate indicator virulence capacity.

Due to differences in 16SrRNA sequence analyses, Salmonella enterica (2443 serotypes) and Salmonella bongori are currently considered to be separate species (20 serotypes). Warm-blooded animals are the primary source of S. enterica isolation, while cold-blooded animals are the primary source of S. bongori isolation.

In addition to being an endemic disease in the tropics and subtropics, typhoid fever has become a significant global public health issue in underdeveloped nations. In developing nations where resistance is rare, antibiotics like ampicillin, chloramphenicol, amoxicillin, and ciprofloxacin are frequently used to treat typhoid fever. However, as the prevalence of diagnosis has increased and drug abuse of the first-line treatment has increased, resistant strains of S. typhi have been chosen. Ampicillin, sulfamethoxazole, and streptomycin resistance is currently widespread.

REQUIREMENTS

 S. no.
1

Salmonella culture

2 Beef/Peptone/Yeast extract agar
3 NaCl solution (0.85%)
4 Centrifuge
5 Glass beads
6 Polyethylene glycol
7 Sodium acetic acid buffer (0.05M)
8 Sephadex G column
9 Dialysis membrane
10 Tris-HCL buffer (50mM, 7.5 pH)
11 MgCl2
12 Bovine serum albumin
13 RNase
14 DNase
15 Trypsin
16 Proteinase K
17 Acetone
18 Chloroform-Methanol solution
19 Distilled water
20 Phenol
21 SDS-PAGE

PROCEDURE

Isolation of O & H Antigen from Salmonella typhi

Flagella Isolation (H antigen)

  1. Grow salmonella culture on beef extract agar for 24 hr at 37˚c.2
  2. Add culture to 0.85% NaCl and centrifuge in cold condition at 5000rpm for 30min.1
  3. Resuspend the pellet in 0.85% NaCl and centrifuge at high rpm with glass beads for 1hr.
  4. Collect supernatant and recentrifuged in cold condition for 5000rpm for 30min.
  5. Take pellet and concentrate with polyethylene glycol.
  6. Dialyzed the mixture against 0.05M sodium acetic acid for 1 day.
  7. Add dialyzed mixture to Sephadex G column and add 0.05M sodium acetic acid buffer (5.8 pH) for purification
  8. Collect fractionation and concentrate with PEG, and kept at 4˚C

Isolation of O & H Antigen from Salmonella typhi

LPS Isolation (O antigen)

  1. Add grown culture to 100ml 50mM Tris-HCL buffer (7.5 pH).
  2. Add 0.2g MgCl2, 0.5 mg bovine serum albumin, 100µl RNase and 100µl DNase.1
  3. Mix the above mixture properly and kept at 60-75˚c for 10 min.
  4. Add 100µl Trypsin and incubate at room temperature for 1hr.
  5. Add 100µl proteinase K, shake well and incubate at room temperature for overnight.1
  6. Centrifuge the mixture at 10,000rpm for 1 hr.
  7. Collect pellet and add 100ml acetone, mix well and centrifuge it at 5000 rpm for 15min.
  8. Collect pellet, add chloroform-methanol solution for overnight at room temperature.
  9. Centrifuge the mixture at 5000rpm for 20 min.
  10. The pellet resuspended in 50 ml distilled water and extracted with equal volume of aq. phenol for 20 min with shaking.
  11. The mixture were cooled on ice and centrifuge at 5000rpm for 20 min.
  12. The phenol phase re-extracted with water, the aqueous phase evaporated and dialyzed against distilled water for 1-2 days and kept at 4˚c.

CONCLUSION

The H and O antigen isolated successfully from salmonella sp. Culture and conform it by using SDS-PAGE gel electrophoresis. For H antigen band visualization use commasie brilliant blue dye and for O antigen use silver stain.

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REFERENCES

  1. Ayse Nalbantsoy, Ismail Karaboz, Radka Ivanova, Ismet Deliloglu-Gurhan, “Isolation and purification of O and H antigens from Salmonella Enteritidis as diagnostic tool”, Ann Microbiol, 2010, 60:565-571.
  2. H. FUJITA, S. YAMAGUCHI, “Studies on H-0 Variants in Salmonella in Relation to Phase Variation”, Journal of General Microbiology, 1973, 76, 127-134.
  3. Oluyege A.O, Babalola J. A, Igbalajobi A.O, Oloruntuyi A.B, “Isolation and Characterization of Salmonella typhi from Widal Positive Patients Attending Ekiti State University Teaching Hospital”, International journal of current microbiology and applied sciences, 2015, vol. 4(10):774-784.
  4. Rucha Patki, Sunil Lilani, Dhaneshwar Lanjewar, “Baseline Antibody Titre against Salmonella enterica in Healthy Population of Mumbai, Maharashtra, India”, International journal of Microbiology, 2017.

Also read:

FAQs

Q.1   Any other method for conformation of isolated antigen?

Ans.  Yes, instead of gel electrophoresis you can use ELISA.

Q.2   There is any analytical methods for purity and conformation checking?

Ans.  Yes, there is use of analytical methods like Bradford assay, Lowry assay, thiobarbiuric acid assay etc.

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