Until now, the production of germ-free birds was limited to animal breed for research. We show here that it is possible to do it with commercial animals. This technique makes it possible to carry out studies of the influence of the microbiota using the genetic background of farm birds.
When attempting this protocol, it is important to control the breeding of the chosen species in the standard conditions and to possess strong knowledge of working in isolators. Begin by setting up 50 milliliter tubes, plastic pipettes, irradiated feed, autoclaved water and sterile sealed plastic containers in a rigid insulator under positive pressure. Fill the transfer germicidal trap with a 2%quaternary ammonium solution and sterilize the isolator three times with formaldehyde vapor moving the materials inside the isolator between each sterilization to ensure the disinfection of all contact surfaces.
At least two days before introducing the eggs, set the isolator temperature to 37 degrees celsius. On the day of introduction, set the hydrometer to 65 to 70%of relative humidity. After selecting clean and flawless eggs, immediately decontaminate the egg surface by dipping them in a 1.5%peracetic acid solution for five minutes, then transport the eggs to the experimental facility using a box decontaminated with formaldehyde vapor.
Store the eggs for 24 hours at 14 degrees celsius, then repeat the sterilization with 1.5%peracetic acid and place the eggs in a hatching incubator for 19 days. On day 19, verify the fertility, viability and development of the embryo using a light egg candler under sterile conditions. Select live, embryonated eggs and decontaminate them by spraying them with a 1.5%peracetic acid solution for 30 seconds or until the entire surface of each egg is covered.
Transfer the eggs into the sterile hatching isolators and rinse them with sterile demineralized water before placing them in the hatching space. After hatching, the animals are kept in the sterile isolators, fed ad libitum with a commercial diet sterilized by gamma irradiation and watered with autoclaved tap water. One day after hatching, take a fecal sample directly from the rectum of different chicks and pool them in a sterilized glass tube.
Add one milliliter of stool to nine milliliters of thioglycolate broth with resazurin and add the remaining sample to nine milliliters of Brain Heart Infusion or BHI broth. Incubate the tubes at 37 degrees celsius without shaking for 18 to 48 hours. At 18 hours, visually examine the tubes for any modification in the growth media.
After 48 hours, take a drop from the BHI fecal broth media, place it on a glass slide and observe it under a microscope. If presence of bacteria is suspected, take a sample from the BHI culture and seed it onto a BHI agar plate, then incubate the plate at 37 degrees for 18 to 48 hours. If colonies appear, identify the bacteria using a high-throughput technique such as MALDI-TOF mass spectrometry.
Six runs of germ-free chicks generation were conducted with ROSS PM3 eggs coming from two different French farms. A total of 853 eggs were collected and after two decontamination steps and 19 days of incubation, 86.40%were viable. Out of the viable eggs, 490 underwent a third decontamination step and were introduced into various hatching isolators for an average hatching rate of 79.80%This represents a hatching rate of 68.94%compared to the initial number of eggs collected.
Hatching results vary substantially from 41.67%to 88.16%of viable chicks compared to the number of eggs collected. This effect was directly correlated with the age of the laying hens where eggs coming from older hens were less viable. The six runs were carried out using four different hatching isolators.
After bacteriological control, the animals from 14 of the 16 isolators were confirmed to be germ free, corresponding to a success rate of 86.5%The key point of this procedure is to maintain the sterility of the isolator. This requires compliance with disinfection procedures and above all, controlling the introduction of the elements via the liquid airlock. This procedure provides animals to measure the impact of the microbiota on the physiology and health of birds.
It can be adapted and used to study microbiota from other farmed birds or wildlife if the eggs are accessible.
