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Evaluation of a Salmonella Strain Isolated from Honeybee Gut as a Potential Live Oral Vaccine Against Lethal Infection of Salmonella Typhimurium

Polish Journal of Microbiology
Volume 68 (2019): Issue 2 (June 2019)
By:
Open Access
|May 2019

Figures & Tables

Fig. 1.

Amplicons of 1.4 kbp are visible for each Salmonella isolate.According to literature, the universal primer (27F, 1492R) gives a PCR product of 1.4 kbp or 1.5 kpb. M is the marker used of 4 kbp size.
Amplicons of 1.4 kbp are visible for each Salmonella isolate.According to literature, the universal primer (27F, 1492R) gives a PCR product of 1.4 kbp or 1.5 kpb. M is the marker used of 4 kbp size.

Fig. 2.

The phylogenetic tree constructed by MEGA X, with the 16S rRNA sequences of the 10 Salmonella strains and two out-groups of Salmonella bongori. The UPGMA method was used with 2000 bootstrap replications, bootstrap percentages are indicated on nodes. The ten Salmonella species in this study are denoted as previously in this study along with their NCBI accession numbers. Evolutionary distances were computed using the Maximum Composite Likelihood method and they are provided in the units of number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2). All positions containing gaps and missing data were eliminated. There were a total of 1034 positions in the final dataset.
The phylogenetic tree constructed by MEGA X, with the 16S rRNA sequences of the 10 Salmonella strains and two out-groups of Salmonella bongori. The UPGMA method was used with 2000 bootstrap replications, bootstrap percentages are indicated on nodes. The ten Salmonella species in this study are denoted as previously in this study along with their NCBI accession numbers. Evolutionary distances were computed using the Maximum Composite Likelihood method and they are provided in the units of number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2). All positions containing gaps and missing data were eliminated. There were a total of 1034 positions in the final dataset.

Fig. 3.

Geometric mean titers of the antiserum against ten different strains.Bars indicate the GMT (± SEM) at 95% Confidence Interval (CI) (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).
Geometric mean titers of the antiserum against ten different strains.Bars indicate the GMT (± SEM) at 95% Confidence Interval (CI) (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Fig. 4.

Comparative indirect hemagglutination titers GMT in the three vaccinated groups and the control group during the 70-days animal trial.
Comparative indirect hemagglutination titers GMT in the three vaccinated groups and the control group during the 70-days animal trial.

Fig. 5.

Survival rate of four groups of rabbits immunized. All four groups were subjected to LD50 of S. Typhimurium. The three groups (R1, R2, and R3), immunized with different doses of HB1 live antigen, showed a significance increase (p < 0.0001) in the survival time in comparison to the control group (R4). Each group consisted of 10 rabbits. Kaplan Meier graph was generated by GraphPad Prism 7.
Survival rate of four groups of rabbits immunized. All four groups were subjected to LD50 of S. Typhimurium. The three groups (R1, R2, and R3), immunized with different doses of HB1 live antigen, showed a significance increase (p < 0.0001) in the survival time in comparison to the control group (R4). Each group consisted of 10 rabbits. Kaplan Meier graph was generated by GraphPad Prism 7.

Geomean mean titers of the sera from four group of rabbits immunized with the live HB1 antigen_

Days post vaccinationR1R2R3R4
109.112.112.1–
2017.142.227.8–
3034.290.545.2–
4064119.4122.4–
5073.548.578.7–
6029.827.89.1–
706.685.8–

Details of collection of the fecal/stool/dropping samples along with location of the sampling_

SpeciesSample sizeAverage weight (gm)Location of the sample collection
Human157Allied Hospital, Faisalabad, Pakistan. District Headquarters Hospital, Faisalabad, Pakistan
Cat155Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan
Sheep154Livestock farm, University of Agriculture, Faisalabad, PakistanDepartment of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan
Honey bee150.2Botanical garden, University of Agriculture, Faisalabad, PakistanDepartment of Entomology, University of Agriculture, Faisalabad, Pakistan
Poultry birds153Poultry farms, University of Agriculture, Faisalabad, Pakistan
Horse158Brooke Hospital, University of Agriculture, Faisalabad, Pakistan

Results of biochemical characterization by Remel RapIDTM and the analysis by ERIC software_

Salmonella isolateRapID No.Percentage IDSpp. identification
TY1032041099.45%Salmonella I
TY2032041099.45%Salmonella I
TY3032041099.45%Salmonella I
HB1236041099.9%Salmonella I
HB11636321099.9%Salmonella I
SP6636040099.9%Salmonella I
CT7616040075.5%Salmonella I
PB2230001098.10%Salmonella I
PB9230001098.10%Salmonella I
HE13636040099.9%Salmonella I
S. Typhimurium ATCC 14028636041099.9%Salmonella I

The immunological cross-reactivity assay for the Salmonella strains with homologous and heterologous antibody titers and geometric mean titers_

Antiserum
AntigenTY1TY2TY3HB1HB11SP6CT7PB2PB9HE13
TY11:641:321:161:161:161:21:41:21:41:8
TY21:161:641:321:161:321:41:81:41:21:4
TY31:161:321:641:81:161:41:21:21:41:2
HB11:321:161:161:1281:321:161:81:161:81:16
HB111:161:81:161:321:641:81:41:41:81:4
SP61:41:21:41:161:81:321:21:81:41:2
CT71:41:21:81:81:161:41:641:81:41:8
PB21:81:41:81:161:81:161:81:641:161:8
PB91:41:21:81:161:41:21:41:81:321:8
HE131:81:141:81:161:41:41:41:41:21:64
GMT11.3181318.3813.936.0635.6576.4985.6576.964

Schedule of Salmonella antigen inoculation for the raising of antibodies against Salmonella strains_

Salmonella antigenDay 1 = 1st shot (0.2 ml)Day 7 = 1st booster (0.5 ml)Day 14th = 2nd booster (0.5 ml)
HR1CHR1CHR1C
TY10.20.20.50.50.50.5
TY20.20.20.50.50.50.5
TY30.20.20.50.50.50.5
HB10.20.20.50.50.50.5
HB110.20.20.50.50.50.5
SP60.20.20.50.50.50.5
CT70.20.20.50.50.50.5
PB20.20.20.50.50.50.5
PB90.20.20.50.50.50.5
HE130.20.20.50.50.50.5
DOI: https://doi.org/10.33073/pjm-2019-017 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
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Language: English
Page range: 173 - 183
Submitted on: Oct 9, 2018
Accepted on: Jan 19, 2019
Published on: May 31, 2019
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
antigen,
cross-reactivity,
16S rRNA gene,
geometric mean titer
Related subjects:
Life sciences,
Microbiology and virology

© 2019 HASSAN ZAFAR, SAJJAD UR RAHMAN, SULTAN ALI, MUHAMMAD TARIQ JAVED, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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