In the present study, effect of agitation rate on growth yield and virulence property of Bordetella pertussis (BP) strain 509 during pilot scale cultivation was investigated. The results indicated significant decrease in growth yield on increasing the agitation rate due to shear stress of bacterial cells. To characterize the shear stress on bacterial cell protein FHA, cell lines such as Vero cells and CHO cells were used to check the adherence property. Finally the virulence property of vaccine culture was screened by Mouse Weight Gain Test (MWGT) and lymphocytosis promoting factor (LPF) test using LACA strains of laboratory mice weighing 14-16 grams. Comparatively more toxicity was observed on higher agitation rates than in optimal and lower agitation. It is evident that higher agitation causes shearing stress in cells inducing more toxicity than optimal agitation which in turn affects the quality of final vaccine.
Table of Contents
Introduction
Material and Methods
Preparation of Media
Opacity Test
Sterility Test
Effect of Agitation
Mouse Weight Gain Test
LPF Activity
Filamentous Haemagglutinin (FHA) Adherence Activity towards Vero & CHO Cell Line
Results and Discussion
Conclusion
Research Objectives and Key Themes
The primary objective of this study is to investigate the impact of various agitation rates on the growth yield and virulence properties of Bordetella pertussis strain 509 during lab-scale cultivation to optimize vaccine production quality.
- Optimization of agitation rates in bioreactors
- Evaluation of shear stress on bacterial cell viability
- Assessment of vaccine toxicity via Mouse Weight Gain Tests (MWGT)
- Analysis of filamentous haemagglutinin (FHA) adherence properties
- Determination of Lymphocytosis Promoting Factor (LPF) activity
Excerpt from the Publication
Effect of Agitation
In order to evaluate whether shear forces affect cell-bound FHA and other cellular factors involved in protection, B. pertussis strain 509 was grown in a 23 liter bioreactor having a working volume of 20 liter, operated at different levels of shear stress as mentioned above. Bacterial cells cultured for 24 hrs under each condition were centrifuged at 8000 g for 15 min, washed and assayed to determine their haemagglutinin activity (HAT) and degree of adherence to Vero and CHO cells [10]. Briefly, 1-day-old Vero and CHO monolayer cells were prepared in a small chamber containing 1 ml of fresh Minimum Essential Medium (MEM). One-tenth volume of B. pertussis cell suspension was added to monolayer cells, mixed gently and allowed to settle for 2 hrs in a 5% CO2 incubator at 370C. The monolayer cells were then washed thoroughly with MEM, fixed with methanol-acetic acid (3:1 ratio) for 10 min and stained with Hoechst No. 33258 solution (2'-[4-hydroxyphenyl]-5- [4-methyl- 1- piperazinyl]- 2,5'-bi-lH-benzimidazole, Sigma, St Louis, MO, USA). Adherent bacteria were counted by fluorescence microscopy of randomly-selected fields showing 3-5 Vero and CHO cells per field. The value presented for each assay is the mean of three standardized experiments. The significance of the differences between the mean values of each condition was determined with the Least Significant Difference (LSD) test at a confidence level of 95%.
Summary of Chapters
Introduction: Provides background on the role of vaccines in preventing pertussis and describes the biological characteristics and pathogenicity of Bordetella pertussis.
Material and Methods: Details the experimental procedures, including medium preparation, cultivation parameters, sterility checks, and specific toxicity testing protocols such as MWGT and LPF assays.
Results and Discussion: Presents and analyzes the experimental data regarding growth yields, pH levels, and toxicity tests under different agitation speeds, highlighting the correlation between shear stress and vaccine quality.
Conclusion: Summarizes the findings by recommending an optimal agitation rate of 140 rpm for producing high-quality, non-toxic vaccine cultures while discouraging higher speeds due to cell lysis.
Keywords
Agitation rate, Bordetella pertussis, Endotoxin, Toxicity, Vaccine production, Shear stress, Growth yield, Mouse Weight Gain Test, LPF, FHA, Cell adherence, Bioreactor, Virulence, Immunogenicity, Cultivation.
Frequently Asked Questions
What is the core focus of this research?
The study examines how different agitation rates during the cultivation of Bordetella pertussis influence the yield and the virulence profile of the resulting vaccine.
What are the primary themes addressed in the paper?
The paper covers cultivation parameters, shear stress analysis, vaccine toxicity testing, and the functional adherence properties of bacterial cells.
What is the main research question?
The researchers sought to determine the optimal agitation rate for B. pertussis cultivation that maximizes growth yield while minimizing the toxicity caused by cell lysis.
Which scientific methods were employed?
The study utilized standard microbiological culture techniques, Mouse Weight Gain Tests (MWGT), LPF activity assays, and fluorescence microscopy to evaluate cell adherence.
What topics are discussed in the main body?
The main body details the preparation of media, the effect of mechanical shear on bacteria, the evaluation of vaccine purity, and the correlation between agitation speeds and animal test results.
How can this work be described using keywords?
Key terms include agitation rate, Bordetella pertussis, endotoxin, toxicity, vaccine production, shear stress, and cell adherence.
Why is higher agitation considered detrimental to vaccine quality?
Higher agitation causes mechanical shear stress, leading to cell lysis and the release of endotoxins, which increase the toxicity of the vaccine.
What significance does the Mouse Weight Gain Test have in this study?
The MWGT is used as a regulatory benchmark to ensure the vaccine meets safety standards; failure to gain sufficient weight indicates the culture is too toxic for use.
How does shear stress affect the adherence of FHA cells?
Shear stress damages the Filamentous Haemagglutinin (FHA) protein on the surface of the bacteria, significantly reducing its ability to adhere to target cell lines like Vero and CHO.
- Arbeit zitieren
- Vidhya Charan (Autor:in), 2011, Effect of Agitation Rate on Growth Yield and Virulence of Bordetella pertussis Strain 509 in Lab Scale Cultivation , München, GRIN Verlag, https://www.grin.com/document/176400
