Growth of Bacterial Strains
In this study, STEC isolates (H2- stx1 and stx2 positive) were grown well and identified by developing white and metallic greenish color on the surface of the TSA and EMB medium as shown in Fig. 1A and B, respectively. On TSB broth medium OD600 of 1.03 STEC strain broth culture was obtained Fig. 1C, which was used for extraction of LPS.
Purificationof Lipopolysaccharide O antigen of STEC strain using chloroform-methanol method
For pAb serum against STEC strains production, above 2ml of diluted immunogen (LPS) of STEC strain was extracted and purified from around 250 ml of overnight broth culture of STEC strainusing modified chloroform-methanol method as shown in Fig. 2. However, the concentration of the extracted and purified LPS was not determined and known.
Determination of the Purity of Purified LPS by SDS-PAGEFollowed by Coomassie Blue Staining
SDS-PAGE separation and coomassie blue staining of the LPS preparation was used to detect and visually characterized the purified LPS. The purified LPS was visualized by direct staining following separation on SDS-PAGE using Coomassie blue staining;as shown in Fig. 3, the purification procedure has eliminated most of the cytoplasmic and membrane proteins with the exception of some protein at the size of BSA indicated on the gel. However, LPS on the gel could not be stained with Coomasie blue since it is not an appropriate stain; due to the difficulty of getting silver nitrate at local market, which could stain LPS on the gel, LPS could be visualized on the gel.
Production of Anti- STEC Strain LPS Mouse Polyclonal Antibodies
In this study for pAb production, among the 40(30 for treatment and 10 for negative control) female mice 10(25%) mice from experimental and 2(20%) mice from control were died. From the died 10 experimental mice, 3 mice were died before immunization during acclimatization time (within 7 days). While, the remaining 7 mice died after the 1st immunization with LPS of STEC strain with alum adjuvant. Before immunization above 2ml of whole blood was collected from 37 mice Fig. 4A and from this 0.5ml of serum was obtained Fig. 4C and used as negative control serum during immunodiagnostic assay development in this study. After immunization of the mice with LPS of STEC at the 1st, 2nd, and 3rd time, on average 0.02 ml, 0.025ml and 0.05ml of serum (pAb) against LPS of STEC strain were obtained Fig. 4D from each mice, respectively.
Slide Agglutination Immunodiagnostic Assay Development for STEC Strain Detection
Slide immunodiagnostic assay was developed for the detection of STEC strains by using anti-LPS of STEC strain pAb serum produced by in this study. Typical agglutination reactions were obtained within 1-2 minutes of mixing the antibody with bacterial as well as purified LPS. The designed diagnostic assay in this research was tested by following the method described in materials and methods, serum from pre-immunized (as negative control), 1ry immunized (as primary response) and 2ryimmunized (as secondary response) and 3ry immunized (as tertiary response) with LPS of STEC strain were checked. The result showed as indicated in Fig.5A no agglutination, B low a gglutination, C medium agglutination and D high agglutination, respectively, were generated. The strength and visibility of agglutination positive result of tertiary immune response test Fig. 5D, was higher than primary Fig. 5B and secondary immune response Fig. 5C.
In addition, the performance of the developed assay was assessed for the comparative detection of LPS purified and colony of STEC strain; in both types of antigen preparations, a representative agglutination result was generated within 1 to 2 minutes. As shown in the Fig. 6, the designed slide agglutination assay in this study was able to detect both LPS and overnight grown colony of STEC strains. However, colony from the overnight culture of STEC has shown strong agglutination result than with their LPS.
Comparison of Appropriate BacterialCulture Medium for Performing Agglutination Assay
The appropriate culture medium for performing slide agglutination assay for STEC strain detection was determined following procedure described in material and methods. Accordingly, the results showed that overnight colony from EMB Fig. 7A, TSA Fig. 7B and broth culture from TSB Fig. 7C, showed medium, high and low level of agglutination, respectively. Thus, bacterial colonies taken from overnight culture on TSA will be the best samples for performing the assay.
Evaluation of the Specificity of the Designed Slide Immunodiagnostic Assay to Detect STEC Strain
The specificity of the newly designed diagnostic assay was determined using 10 STEC, 3 EHEC, 6 non-diarrheic E.coli, 9 EPEC, 4 non E. coli (Klebsellapneumoniae, Shigellaflexinari, and SalmonellaTyphimurium and Staphylococcus aureous) samples. The test results showed (Table1) that the designed immunodiagnostic assay produced in this study performed very well. It was found that 10 (100%) of 10 STEC strains showed positive agglutination with the pAb against STEC strain; 7 (77.8%) of 9 EPEC strains showed negative agglutination with the pAb serum against STEC strain and 2(22.2%) showed positive agglutination. Furthermore, 6 (100%) of 6 E. coli isolates from non-diarrheic animals showed no agglutination, 2 (66%) of 3 EHEC strains showed positive agglutination and 1 showed negative agglutination with the pAb serum against STEC strain. In addition, 4 (100%) of 4 non E. coli spp showed negative agglutination with the pAb serum against STEC strain. As showed in (Table 1) and data analysisin (Table 2) the specificity of the newly designed immunodiagnostic assay was approximately 89.5% by using the formula of TN/ (TN + FP) × 100%. The cross-reactivity (non specific agglutination) of the generated antiserum (pAb) was shown in only two (10.5%) of the 19 strains of non-STEC and non-E. coli bacteria strains included in this study.
Table 1
Specificity of the slide agglutination assay designed for STEC strain detection
Bacterial strain type
|
ID
|
Agglutination reaction
|
Remark
|
STEC strains
|
H2
|
+
|
|
D43
|
+
|
|
D19
|
+
|
|
D27
|
+
|
|
D24
|
+
|
|
D29
|
+
|
|
D23
|
+
|
|
D8
|
+
|
|
D7
|
+
|
|
H1
|
+
|
|
EHEC strains
|
N1O7
|
+
|
|
N166
|
-
|
|
N98
|
+
|
|
EPEC strains
|
K51
|
-
|
|
D33
|
-
|
|
K33
|
-
|
|
39
|
-
|
|
38
|
-
|
|
16
|
+
|
|
56
|
-
|
|
H16
|
+
|
|
43
|
-
|
|
Non-diarrheic strains
|
N158
|
-
|
|
N154
|
-
|
|
N159
|
-
|
|
N157
|
-
|
|
N156
|
-
|
|
N155
|
-
|
|
Non-E. Coli strains
|
Klebsella pneumonia
|
-
|
|
Shigellaflexinari
|
-
|
|
Salmonella Typhimurium
|
-
|
|
Staphylococcus aureous
|
-
|
|
‘+’ Positive agglutination;‘–‘ Negative agglutination
As indicated in the Table 2, True Positive (TP), True Negative (TN), False Positive (FP) and False Negative (FN) data were generated from the test result of immunodiagnostic assay developed by in this study. The generated data were analyzed using MedCalc Version 18.11.3 Software packages and the sensitivity, specificity, positive predicted value, negative predicted value and accuracy of the developed immunodiagnostic assay kit was determined as followes:
Table 2
Statistical data analysis using MedCalc Version 18.11.3 Software
Data Entry for Diagnostic Test Evalution for STEC Strain Detection
|
Test
|
Present (STEC)
|
No
|
Absent(STEC)
|
No
|
Total
|
Positive
|
TP
|
12
|
FP
|
2
|
14
|
Negative
|
FN
|
1
|
TN
|
17
|
18
|
Total
|
-
|
13
|
-
|
19
|
32
|
Detection efficiency result of the developed diagnostic assay for STEC detection
|
Statistic
|
Formula
|
Value
|
95%CI
|
Specificity
|
(TN/(TN+FP))100%
|
89.5%
|
66.86 to 98.7%
|
Sensitivity
|
(TP/(TP+FN))100%
|
92.31%
|
63.97 to 99.81%
|
Positive predicted value
|
(TP/(TP+FP))100%
|
85.7%
|
61.57 to 95.74%
|
Negative predicted value
|
(TN/(TN+FN))100%
|
94.4%
|
71.99 to 99.12%
|
Accuracy
|
(TP+TN)/(TP+TN+FP+FN) 100%
|
90.6%
|
74.98 to 98.02%
|
|
|
|
|
|
|
|
|
|
Determining the Sensitivity of a Slide Immunodiagnostic Assay to Detect STEC Strains
The sensitivity of the slide agglutination assay developed in this study could detect STEC strains at a concentration of ≥12,400 X105CFU/ml using overnight colony of STEC Fig. 9 and Table 3. Based on the results obtained the sensitivity of the assay was approximately 92.3% as shown inTable 2.
Table 3
Sensitivity of the slide agglutination test assay for STEC strain at the indicated dilution (fold)
‘+’ Positive agglutination; ‘–‘Negative agglutination
From the serially diluted colony of STEC strain the slide agglutination test showed positive result up to 10-5 dilution. From this 5 µL of aliquot was transferred in to TSA medium for counting colony forming unit and 62 colonies were grown in 5 µL colony dilutions Fig. 9B, which means 12,400 X105 CFU ml-1.
Evaluating the Detection Capacity of the Designed Assay in Human Feces Spiked with STEC Strain
To determine if the developed slide immunodiagnostic assay could be used for detection of STEC strain in fecal samples, human feces were collected and spiked with pellet part of TSB broth STEC culture Fig. 10. The spiked sample was then tested on slide agglutination assay as described in material and methods, the result indicates that the slide agglutination assay developed in this study could detect spiked (STEC in feces) sample at a concentrations of approximately up to 9, 600 X103 CFU ml-1 Fig.11.
In addition, the spiked sample was serially diluted with PBS from 100 to 10-9 dilution factors. From the serially diluted colony of STEC strain the slide agglutination test showed positive result up to 10-3 dilution. From this 5 µL of aliquot was transferred into TSA medium for colony forming unit counting; thus, 48 colonies were grown /5 µL diluted sample Fig. 13, which means this diagnostic assay could detect at a concentration of ≥ 9,600 X 103CFU ml-1STEC from fecal sample Fig. 12.