Summary
Virulence indicates the strength of a pathogen's ability to cause disease, and the substances that make up bacterial virulence are called virulence factors, mainly invasiveness (the ability of pathogenic bacteria to colonize the body, break through the body's defense barriers and internalize, reproduce and spread) and toxins.
The assessment of pathogenic bacterial virulence is often carried out by means of infection models, and common infection models include in vitro models (cellular infection models) and in vivo models (animal infection models); among the non-vertebrate infection models, insects have their complex innate immune system, which is highly similar to that of mammals, and they have been considered to be a suitable alternative model for the study of bacterial virulence in large mammals.
Principle
The larvae of Galleria mellonella are excellent model organisms for in vivo virulence testing of pathogens. Intravitreal injection through the left forefoot or skin is the most common route of infection, and a health scoring system based on the mobility, melanization, and survival of the larvae of Galleria mellonella can be established for evaluating the virulence of the pathogens to be tested.
Operation method
Pathogen virulence determination
Principle
The larvae of Galleria mellonella are excellent model organisms for in vivo virulence testing of pathogens. Intravitreal injection through the left forefoot or skin is the most common route of infection, and a health scoring system based on the mobility, melanization, and survival of the larvae of Galleria mellonella can be established for evaluating the virulence of the pathogens to be tested.
Materials and Instruments
Large wax borer larvae, strain to be tested, bacterial liquid medium, bacterial solid culture plate, sterile PBS, visible spectrophotometer, sterilized microfeeding needle. Move The steps for virulence determination of pathogenic bacteria are as follows: 1、Bacterial recovery: Take the common pathogen Escherichia coli as an example, the strain to be tested was recovered in the form of zoned lines on MH solid medium overnight culture; the next day, a single colony was randomly picked and inoculated into 2 mL of MHB liquid medium, placed in 37 ℃ shaking bed, 200 rpm shaking bacteria overnight; 2、Bacterial culture: take the bacteria shaken overnight 1:100 inoculated in fresh MHB liquid medium, placed in 37 ℃ shaking bed, 200 rpm shaking to logarithmic growth period; 3. Measure the absorbance ( OD600 ) at 600 nm of the bacteria in the logarithmic growth stage in step 2 using an enzyme meter or a spectrophotometer; 4. Centrifuge the bacterial solution at 5 000 rpm for 5 min, discard the supernatant, and then resuspend the bacteria with PBS and adjust the OD600=1.0 (about 5 × 108 CFUs/mL); 5. Randomly group the larvae of the wax borer into 10 petri dishes, and make sure that the length, weight and growth status of the larvae are the same as far as possible; 6、Use PBS to dilute the bacterial solution to be tested to 1 × 107 CFU/mL. fix the larvae, expose the abdomen, and use a microsyringe to inject 10 μL of the diluted bacterial solution (the bacterial infection quantity is 105 CFU) into the left anterior first gastropod, while the control group was injected with the same amount of PBS; 7、Place the larvae of the wax borer in different petri dishes in a constant temperature incubator, incubate them at 37 ℃ under light protection, observe them once every 12 hours for 72 hours, record the survival of the larvae of the wax borer and draw the survival curve; 8. Infection tests with different orders of bacteria can be carried out, and the Probit model is used to make a function curve between the bacterial content and the death situation, with the x-axis being the bacterial content and the y-axis being the proportion of deaths of the larvae of the wax borer. According to the fitted function curve, the x-value corresponding to the value 0.5 on the y-axis is the LD50. Caveat 1. The growth and development of the larvae of the emerald ash borer (EAB) have strict requirements on temperature, so they can be stored at 15 ℃, and then reared at a constant temperature of 37 ℃ after infection;2. starvation can reduce the immune response and enhance the susceptibility of the emerald ash borer, thus increasing the chance of successful infection, therefore, the emerald ash borer larvae were usually starved for 24 h before infection to facilitate the successful establishment of the infection model;3. Intravital injection through the left forefoot or skin is the most common route of infection. Oral infection has also been reported, with the disadvantage that it is difficult to control the exact infective dose, which can be solved by force-feeding method, but it is challenging to the operation technique;4. The need to specify in advance the bacterial OD600The corresponding relationship between bacterial OD 600 and CFU should be clarified in advance, the specific method is as follows: dilute the bacterial solution to OD600= 1.0, and gradient dilution and plate counting. Common Problems 1. How to determine the infective dose and observation time for pathogenic bacteria? It is recommended to refer to the literature, the infective dose required for bacteria with different virulence backgrounds varies greatly, and a gradient pre-test between 104~106 CFU can be performed to determine the optimal bacterial infective dose and observation time interval. 2. How is the control group set up during the experiment? In addition to using PBS injection as a negative control, high and low virulence strains of the same genus of bacteria can be selected as a control according to the category of the strain to be tested in order to assess the virulence strength of the strain to be tested. For more product details, please visit Aladdin Scientific website.