马达加斯加嘘声蟑螂作为非哺乳动物的替代动物模型来研究毒力、致病机制和药效

The overall goal of this work is to assess the effects of bacterial infection in a novel surrogate animal model, the insect, Madagascar Hissing Cockroach, from the genus Gromphadorhina. The use of the Madagascar Hissing Cockroach as an alternative animal model, can be extraordinarily helpful in answering questions in microbiology. These include studies of the host-pathogen interactions and microbial pathogenesis.

Specifically, it can be extraordinarily helpful in the identification of virulence factors that are common to different animals. In addition, such a model, the Madagascar Hissing Cockroach, can be helpful in identifying drugs that are effective in the treatment of infections. Large screening studies to look for attenuating mutations in different pathogens, can easily be done using this animal model.

The hissing cockroaches are also easy to obtain, to handle and care for, and the cost associated with their use is low. Though this method has provided insight into Burkholderia pathogenesis, it also could potentially be applied towards the study of other bacterial pathogens. The use of the hissing cockroach first came to us when we encountered technical and logistical difficulties using the Galleria waxworm model at mammalian temperatures.

A former intern, who also volunteered at a nature center that had plenty of hissing cockroaches, suggested that we try them. We did and they worked beautifully. Visual demonstration of this method is critical as the cockroaches are hard to immobilize using one hand while injecting them with inoculum in the other.

Demonstrating the procedures will be Joshua Roan, a student intern in the laboratory. Jared Crumpler, a technician, will also demonstrate an alternative way to immobilize and inject hissing cockroaches. After breeding hissing cockroaches according to the text protocol, transfer the appropriate number of hissing cockroaches in a cage, to a 37 degree Celsius humidified incubator, one to three weeks prior to an experiment for acclimation, to avoid temperature shock.

Check the hissing cockroaches every one to two days to replace high water content food, and to clean the cage. On the day of injection, distribute 6 to 12 acclimatized hissing cockroaches into groups per container, ensuring equal distribution by sex and body mass. Perform all Burkholderia work, including injections, in a class II or a class III biological safety cabinet, in a BSL2 or a BSL3 setting, using institutional recommended personal protective equipment.

Three days prior to infection, prepare a master plate of Burkholderia by streaking bacteria onto LB agar for Burkholderia pseudomallei, or Burkholderia thailandensis, or onto LB agar supplemented with 4%glycerol for Burkholderia mallei. Inoculate 10 to 20 milliliters of LB broth, with several colonies of B.pseudomallei, or B.thailandensis from the master plate. Similarly, inoculate LB broth supplemented with 4%glycerol for B.mallei.

Incubate the broth culture at 37 degrees Celsius and 175 to 250 rpm for approximately 18 hours. Then centrifuge 2 to 3 milliliters of culture at 5, 000 times g for 10 minutes. Discard the supernatant, and resuspend the bacterial pellet in sterile PBS, pH 7.4.

Then use PBS to dilute the bacteria to achieve an optical density at 600 nanometers of 0.5 as measured with a spectrophotometer. Serially dilute the bacteria tenfold in PBS from the starting OD of 0.5, to achieve the needed concentrations of the inoculum in a 25 microliter injected volume. Use these suspensions for infection.

For drug administration, such as chloroquine, dilute the drug of interest to provide the correct dosing per weight of cockroach in the 25 microliter injected volume. For example, chloroquine was diluted to a concentration of 12 milligrams per milliliter to provide 300 micrograms of drug to a 6 gram hissing cockroach. To assemble the injector, set the pointer to the desired volume by rotating the adjustment screw on a repetitive pipette.

Push the release bar of the repetitive pipette inward, and pull the pusher outward. The repetitive pipette is now ready to accommodate a loaded syringe. Attach the syringe to a sterile 26 or 27 gauge half inch, or shorter, needle.

Fill a 1 milliliter syringe with suspension containing either bacteria or drug. Next, tap the syringe to float the air bubbles to the top and expel the bubbles, and some suspension, into a container filled with 10%bleach. Then snap the syringe onto the syringe clip of the repetitive pipette with the needle bevel facing up.

Pull the release bar outward and firmly press the dispenser button to perform blank injections into a container filled with 10%bleach, until the syringe plunger is against the pusher. To carry out injections, use 10%bleach followed by 70%ethanol to clean the work surfaces in the safety cabinet that may come in contact with the hissing cockroaches. Allow the surface to air dry.

To inject a hissing cockroach, with one hand, grip it by its side, and immobilize the insect such that it is unable to recoil during injection. Then bend it slightly so that the cutaneous membranes between the abdominal terga are exposed. For safety reasons, it is necessary to completely immobilize the cockroach so that it is unable to recoil or move during injections.

If a cockroach refuses to remain immobilized, place it back in the cage and return to it after all the cockroaches in the group are injected. Alternatively, hold the cockroach in place in this manner during injections. With the other hand, hold the repetitive pipette such that the needle is at a 0 to 30 degree angle from the dorsoventral midline of the cockroach.

Then puncture the cutaneous membrane adjacent to the third, fourth, or fifth tergum from the posterior end. Entry of the needle into the hissing cockroach at the angle indicated, ensures that the needle does not go through and exit out of the hissing cockroach. The injection site also ensures that the injected material is contained within the abdominal cavity filled with hemolymph.

During injections, great care should be taken to ensure that the needle does not go through the insect. For consistent dosing, or consistent delivery of inoculum between cockroaches, the dispense button on the injector must be firmly pushed each and every time. Firmly push the dispense button to inject the volume.

Then gently withdraw the needle at the same angle as it entered. Place the injected hissing cockroach in a separate container to distinguish it from uninjected insects. Then wipe off any hemolymph that may have oozed out from the injection site.

Use the same syringe and needle to continue injecting additional hissing cockroaches within a group. Stop injecting before the syringe plunger reaches the bottom of the barrel, to prevent incomplete dosing in the last injection. For multiple injections in a single hissing cockroach, such as those with bacteria and drug, inject at different sides of a tergum on the dorsal side of the insect.

Alternatively, perform multiple injections by injecting at different terga. Ensure that the lids of the containers are securely fastened and use tape to reinforce the lids if necessary. After injecting, incubate the hissing cockroaches in a humidified 37 degree Celsius incubator.

To record hissing cockroach morbidity and mortality in a class II or a class III biological safety cabinet, use the following morbidity scoring table once or twice daily, over a period of one to two weeks. Refer to the text protocol for additional details. These survival graphs show that more hissing cockroaches survived in groups that were infected with the attenuated mutants, than in groups that were infected with wild type Burkholderia pseudomallei K96243, parental Burkholderia mallei SR1, or Burkholderia thailandensis DW503.

These graphs illustrate that the hissing cockroach is a viable animal model for both virulent and avirulent species of Burkholderia. Infection with the mammalian avirulent Burkholderia wild type species B.thailandensis E264, and its aminoglycoside-sensitive derivative, DW503, show that the hissing cockroach model is particularly suitable for elucidating mutations in B.thailandensis that lead to attenuation. These graphs demonstrate that increasing concentrations or multiple injections of chloroquine, do not kill the hissing cockroaches, which illustrates that drug toxicity can also be tested in the hissing cockroach model.

In this experiment, hissing cockroaches were infected with B.thailandensis and treated or left untreated with chloroquine, demonstrating the efficacy of chloroquine against B.thailandensis. Finally, this table illustrates how morbidity is scored for the hissing cockroaches. The overall score for a group of hissing cockroaches is based on the animal with the highest score in the group.

Once mastered, the process of injecting 100 hissing cockroaches can be completed in 1 to 2 hours. While attempting this procedure, it is important to remember that practice is the key to becoming proficient, or comfortable, with cockroach injections. We recommend keeping a separate group of cockroaches, just for the purpose of practicing injections.

Following this procedure, other methods, like hemolymph extraction, can be performed in order to answer additional questions, such as the effects of the bacterial infection on the insects'phagocytic cells. Because a hissing cockroach is a viable surrogate host for Burkholderia, we are currently exploring the possibility that it can also be used as an animal model for other Biosafety Level 3 pathogens, such as Francisella tularensis. After watching this video, you should have a good understanding of how we work with Burkholderia and how we perform Madagascar Hissing Cockroach injections.

Don't forget that working with Biosafety Level 3 pathogens can be extremely hazardous. Therefore, always work in biological safety cabinets and use personal protective equipment.