The overall goal of this procedure is to treat amphibians infected with the amphibian Kitt fungus, Bera key tridium, dendro, he or BD using the antifungal chemotherapy at triazole. This is accomplished by first growing BD cultures and inoculating animals with the fungal pathogen. The second step is to test animals using DNA extraction and QPCR on skin swabs to ensure BD infection.
Next, the infected animals are treated by bathing the animals with 0.0025%econazole solution for five minutes a day for six days. The final step is to monitor the infection status of the treated animals by performing QPCR analysis on skin swaps for a minimum of four weeks after the completion of treatment to ensure successful treatment of the animal, this procedure demonstrates that a reduced concentration and shorter treatment time of the antifungal itraconazole is efficient for the treatment of animals infected with bd. The main advantage of using this treatment regimen over the recommended treatment dose, which is a 0.01%itraconazole solution for 11 days, is that a reduced concentration of one quarter of that recommended dose and a reduced treatment time to six days is still effective at curing BD infection, but also minimizes the negative side effects of using the recommended treatment dose.
After incubating the BD in tritone broth as outlined in the text protocol and checking for Zo spore movement, flood each plate with five milliliters of aged tap water and wait for 10 minutes to allow ZO spores to enter the media. Next, pour the Zo Spor suspension into a portable container. Estimate the ZO spor concentration with a hemo cytometer and dilute the mixture to a concentration of one times 10 to the six per three milliliters with age tap water to inoculate an animal such as the Latoria vei Alpina shown here.
Hold it over a 50 milliliter container and pour three milliliters of the inoculum mixture over its venter. Then place the animal into the container with the runoff from the inoculum. Place a lid on the container and leave the animal in the inoculation container for 24 hours to ensure infection.
To prevent contamination with the BD pathogen. Remove the old gloves and place them to be autoclaved prior to disposal. After donning, a new pair of gloves proceed to inoculate the next animal.
After 24 hours of inoculation, return animals to their individual disinfected terraria for two weeks. Remember to disinfect all liquid waste prior to disposal by bringing the solution to 10%bleach after two weeks. Test for successful BD infection by swabbing the animals with a sterile cotton swab.
Gently rotating the swab during and between strokes. Swab five times on the venter on each side, each thigh, and each of the four limbs for a total of 45 strokes. Store the swabs in 1.5 milliliter micro tubes at minus 20 degrees Celsius.
Extract the genomic DNA from the swabs using a commercially available DNA isolation kit and perform QPCR as outlined in the text protocol. Prepare three treatment baths, one for each of the three dosages of itraconazole as described in the text protocol. Next place, individual amphibians in disinfected.
50 milliliter can containers with 35 milliliters of the treatment solution. Remembering to change gloves between animals, gently swirl the container occasionally to ensure that the animals are fully covered for five minutes. After five minutes, return the animals to their disinfected to area.
Repeat the five minute treatment daily for an additional five days. Test the animals again for BD infection one week after treatment finishes and once a week for four weeks to ensure that the infection has cleared. Note that even after treatment has completed, the animals need to be treated as if they're still a contamination risk.
The animals need to be kept in separate to area for at least one month after infection. If the treatment is unsuccessful, it must be repeated. Different species and different life stages respond differently to infection and to treatment regimes, and therefore the treatment must be trialed before its broadly implemented.
As BD is a biohazard disinfect the individual terria weekly by bathing the terria and treatment containers in a 10%bleach solution. Bath for at least 30 seconds, wash away the bleach with two rinses and water and allow the containers to dry for 24 hours before reuse survival of frogs. Following 11 days of BD treatment or six days of BD treatment are shown here.
The black arrows indicate the last day of treatment. The solid lines indicate survival of the control groups. The solid black lines are for animals that were BD positive but did not receive BD treatment.
And the solid gray lines are for animals that were BD negative and did not receive BD treatment. The broken lines show survival of treatment groups receiving the indicated concentration of econazole. All animals treated with 0.0025%in cous nebuliser for six days were BD negative four weeks after treatment ended and thus this treatment was considered successful.
In addition, this dosage of econazole treatment did not differ significantly in mortality when compared to the BD positive controls. Note, the treatments of 0.01%and 0.005%econazole for 11 days resulted in a significant increase in mortality compared to the BD positive controls. Don't forget that the fungal pathogen BD is a biohazard precautions such as wearing and changing gloves, autoclaving, all solid wastes and disinfecting.
All liquid waste must be performed when following this procedure.
