Aeromonads are common contaminants in foods such as fish and other sea foods, raw and cooked meat, poultry, vegetables, milk and milk products. These organisms are responsible for several extraintestinal human infections such as meningitis, endocarditis, peritonitis, osteomyelitis, septic arthritis, septicaemia, eye and urinary tract infections. Keeping the importance of these organisms in mind present study was envisaged to develop immunoassays for efficient and rapid detection of Aeromonas spp. vis-a-vis identification of a common polypeptide moiety in their outer membrane proteins (OMP).Western blot study employing anti-OMP serum indicated polypeptides of 25 and 35 kDa to be immunogenic in most of the Aeromonas strains. The findings of this study will be of immense value in surveillance and monitoring of Aeromonas spp. in foods.
Recent innovation in the isolation of Aeromonas is outer membrane protein based immunoassays. Recent studies on this aspect revealed that outer membrane protein (OMP) profile of Aeromonas spp is specific to certain hybridization groups (Kuijper et al., 1989). However, Stephenson et al., (1987) demonstrated that protein profile differed according to species. Similarly Santos et al., (1996) demonstrated heterogeneity of OMP pattern among motile Aeromonas strains belonging to different sero-groups as well as among the strains of same serotype irrespective of the species and virulence. Merino et al., (1993) developed an ELISA using detector antibody against a surface array protein of A. hydrophila (serotype 0:11) for its detection from food samples. However, this test is not useful for other serogroups of Aeromonas, as the surface array protein used was specific for serotype 0:11. There is a need to identify a protein moiety which is common in all serotypes. This might pave way for the development of an immunoassay for the identification of all Aeromonas serotypes.
Materials and Methods
OMP of 16 Aeromonas strains was separated as per the method of Crosa and Hodges (1981) as modified by Santos et al. (1996). Briefly, Aeromonas grown overnight in 100 ml of tryptic soya broth (TSB, Difco) were reovered by centrifugation at 5000 rpm for 30 min. Cells were resuspended in 3 ml of 10 m mol/l tris buffer containing 0.3% (w/v) ( pH 8.0) and sonicated with a sonifier (MSE, Ultrasonicator) ( 10 amplituted, 45 seconds, 4-5 times). After centrifugation at 10,000 g for 2 min. the supernatant fluids were transferred to new tubes and centrifuged for 1 hr at 17,000 g at 40c. Cell envelop suspensions were incubated with 3% sodium lauroyl sarcosinate ( sarcosyl) (w/v) in 10 m mol/l tris buffer at room temperature for 20 min. Outer memberane protein was obtained by centrifugation at 17000 g for 1 hr and washed twice with distilled water. The OMP was stored at -20oc.
The protein content was determined by the method of Schaterle and Pollack (1973) with little modifications. A small amount (25 μl) of OMP was diluted with 400 μl of distilled water. Different concentrations (25 μg, 50 , 75 , 100 , and 125 ) of bovine serum albumin (BSA) were prepared in DW to a total volume of 400 μl. To all the tubes, 400 μl of 2x lowry concentrate (Appendix ) was added and incubated at room temperature for a minimum of 10 min. Thereafter,200 μl of the 0.2 n Folin reagent (Appendix ) was added and incubated (55 oc for 5 min). The Absorbances were read at 650 nm using polystyrene cuvettes. A standard graph was plotted against optical density (OD) of BSA in different concentrations. Protein concentration of OMP was estimated using standard graph.
Antiserum against OMP of A. hydrophilla (VPH5) was raised in adult rabbits and guinea pigs as per the method described by Santos et al.(1996) with some modifications. OMP (500 μg protein) was emulsified with an equal volume of Freund’s complete adjuvant (FCA,Difco). Rabbits were injected subcutaneously in divided doses at six dorsal sites. Two booster doses were given with OMP (250 μg protein) emulsified with equal volume of Freund’s incomplete adjuvant (IFCA, Difco) at two weeks interval. Rabbits were bled 15 days after the last immunization. Preimmune serum was used as control serum. Guinea pigs were immunized with half of the dose of OMP as usd for rabbits. Rest of the procedure was same.
OMP fractions were analysed by SDS-PAGE as per the method of Laemmli (1970) employing 12.5% (w/v) acrylamide in the resolving gel and 5% (w/v) acrylamide in the stacking gel. The samples were diluted with sample buffer in a ratio of 4:1 and heated at 95ºc for 5 min. Approximately,30μlof the samples containing 100μg protein was loaded in each lane of the gel. The gel was run at 80V for 12 hrs, then stained with coomassie brilliant blue R-250(sigma) staining solution for 8 hrs and finally, destain with destaining solution. Calculation of molecular weight(s) (MW) of the peptide(s) was done by extrapolation of relative mobility of the unknown samples against that of standard molecular weight markers.
Immunoblot of OMP was performed as per the method described by Santos et al., (1996) with little modifications. The polypeptides from SDS-PAGE were transferred to 0.45 μm nitrocellulose paper (Bio-rad) in Electroblot apparatus (Genie, Banglore) using transfer buffer (table 2). After transfer, the membranes were blocked with 0.5% (w/v) gelatine in PBS for 24 hrs at 00C, washed thrice with PBS-T. Membranes were then incubated with anti-OMP serum (1:100 dilution in PBS) at 370C for 1hr. The membranes were again washed with PBS-T and incubated for 1hr with anti-rabbit HRPO conjugate (1:500 in PBS). The reaction was visualized by incubating the membranes with the substrate DAB (Diaminobenzidine) dissolved in PBS (0.5 mg/ml) containing 2μl/ml of H2O2. The reaction was stopped with distilled water.
Results and discussion
OMP were extracted by culturing Aeromonas strains in brain heart infusion broth at 370C with shaking, followed by ultrasonification and centrifugation. Separation of inner and outer membranes was achieved by incubation with sarkosyl. This was important because OMP profiles are reported to be influenced by temperature and air supply to bacterial cultures(Statner et al., 1988). The method followed in this study is reported to give full recovery of OMPs (Kuijper et al.,1989). Outer membrane protein (OMP): OMP of 16 strains was separated and protein concentration estimated (table 1). The protein concentration was found to be in the range of 11.3 mg/ml to 16.2 mg/ml.
SDS-PAGE analysis of OMP of different Aeromonas strains revealed upto 4 major and 4 to 6 minor polypeptide bands. The molecular weight (MW) of the major polypeptide bands, estimated by comparison with standard MW markers run parallel, was in the range of 14 to 55kDa. Large smearing of the bands in the MW range of 40 to 45 kDa was observed. In addition a few minor bands in the lower molecular weight range (10-12 kDa) were also observed in certain strains. Major polypeptides of 14 and 35 kDa and minor polypeptide 25 kDa were common in most of the Aeromonas spp. irrespective of serogroup or species.
Table 1. OMP stains and their concentrations
|Strain no. (OMP)||Protein concentration (mg/ml)|
Heterogeneity in protein profile was observed among Aeromonas strains of different serogroups as well as of same serogroups. However, some similarity was observed for some species of Aeromonas. Protein profile was identical for 2 (F 7 and F 17) A. caviae strains and for 2 (VPH 5 and 10) A. hydrophila starins of rough serogroup.
The SDS –PAGE analysis revealed heterogeneity in protein banding pattern both within and among the species and serotypes, except for a few exceptions. Similar variability in OMP profiles has been described by Santos et al., (1996) who observed that Aeromonas strains belonging to different serogroups and as well as to same serogroup exhibited differences in their protein banding pattern. Aoki and Holland (1985) also observed wide variation in the profiles of A.hydrophila proteins and reported them to be due to serogroup differences, variation in habitat, host range and virulence. They also reported that OMP profile of A.hydrophila differed from A. salmonicida. In an interesting study Kuijper et al., (1989) analysed the OMP of 46 faecal Aeromonas strains from hybridization groups (HGs) 1 (A.hydrophila;n=10),4 (A. caviae, n=16) and 8(A. veronii; n=20) and reported that every isolate of HG-1 and HG-8 had a unique OMP profile, in contrast to isolates of HG-4, which were separated into 5 different OMP types. Our study also revealed some homogeneity among A.hydrophila strains. It may also be pointed out that 2 of the 3 rough strains tested in this study had identical OMP profile. Detailed studies on more number of strains are needed to confirm these findings. The study revealed 4 major and 4 to 6 minor protein bands within the MW range of 14 to 55 kda. In addition some minor bands were also observed in the range of 10 to 12 kDa and 66 kDa with a few strains. This is in agreement to reports of earlier researchers who have noted major protein bands in the range of 30 and 45 kDa (Kuijper et al., 1989). Similarly Santos et al., (1996) observed major proteins to be in the range of 55 and 28 kda. Dooley and Trust (1988) studied the OMP of highly virulence group of strains. They observed that major proteins of MW of 30 kDa and proteins in the molecular weight range of 45 to 55 kDa to be predominant. These variations are not unexpected due to the complex and diverse nature of Aeromonas spp.
Peptides from SDS-PAGE were transferred to nitrocellulose membrane and immunoblots were visualized using rabbit anti-OMP serum as the first antibody and goat antirabbit-HRPO conjugate as the second antibody. Three major immunogenic polypeptides in the MW range of 14,35 and 45 kDa and minor polypeptides of 25 and 66 kDa were observed in homologous
Fig 1. Corresponding western blot analysis of OMP of Aeromonas strains
Aeromonas (VPH 5) strain whereas bands of 25 and 35 kDa in most of the Aeromonas strains were found to be reactive by anti-OMP serum (Fig. 3 & 4). Bands of 45 kDa of few strains (F7, Floor2, 69 and F2) were also immunogenic by anti-OMP serum.
Fig 2. Corresponding western blot analysis of OMP of Aeromonas strains
Large smearing of protein bands in the MW range of 40 to 45 kDa seen in this study may be due to LPS which migrate to the same area of gel as smeared protein (Dooley and Trust 1988) . The present study also revealed that major polypeptides bands of molecular weight of 14 kDa and 35 kDa and a minor peptide of 25 kDa were common in most of the Aeromonas strains. Kuijper et al., 1989 have also observed the presence of 24 to 25 kDa polypeptides in all isolates of HG-4 and HG-8 but not in HG-1. In order to identify imminodominant polypeptides, OMPs of all the strains were subjected to western blotting by employing anti-OMP serum against a rough strain. Use of this antiserum had natural advantage, since as stated earlier, all Aeromonas strains are reported to have rough agglutinins in them. The results indicated that major polypeptides in MW range of 14, 35, 45 and minor polypeptides of 25 and 66 kDa of homologous strain were immunogenic. Of these, polypeptides of 45 kDa were reactive with only a few strains whereas that of 25 and 35 kDa in most of the strains. These common protein moieties probably represent rough proteins in Aeromonas strains. Studies of suitable nature are not available to compare these results. However, in an interesting study Santos et al., (1996) reported that antisera against whole cell of serotypes 03, 06, 011 and 019 reacted with membrane proteins of all the strains of their own serotypes but no to moderate reactions were observed with sera against heterologous serotypes. It appears that there are serotype specific proteins which may not be suitable for diagnostic purposes. Hence it provides indirect evidence that rough protein moiety as observed in present study may be useful tool in developing immunoassays for detection of Aeromonas spp. irrespective of their species and serotypes.
Authors are thankful to Director, Indian Veterinary Research Institute, Izatnagar and Head, Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar for providing the necessary facilities for research.
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