Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

The overall goal of this sampling technique is to reduce the number of fish used for health monitoring and to optimize turnover, cost, and sensitivity of pathogen detection, including when screening imports in quarantine. This technique helps to define the health statutes of the refuge colonies. This method is a useful tool when reading or assessing treatment efficiency.

One main advantage of this technique is the screening and triage of imports in quarantine. It strengthens the biosecurity plan of the aquatic facility. To begin the protocol, set a clean eight liter tank out of a recirculating system.

Fill up the tank with about eight liters of water sourced from sumps. Add two ceramic beads or sponge cubes of biomedia from the systems to screen. Next, add one to do Danio rerio per liter, using wild type fish of the dominant genetic background in the facility, for example A B.Select at least six fish.

Feed the fish once a day. Vary the diets to make sure the sentinels are exposed to all the diets used in the zebrafish facility. To conduct the water change, transfer the sentinels in biomedia into a temporary tank.

Completely empty the sentinel tank and clean it. Refill the sentinel tank with sump water only and place the sentinels and biomedia back. After exposing the sentinels to sump water for four months, euthanize the fish by an approved method such as immersion in an overdose solution of 2-phenoxyethanol.

To confirm death, wait for ten minutes after cessation of operculum movement. Grab the cadaver with forceps and freeze it entirely at negative 80 degrees Celsius in an identified container. Prepare a sterile dry swab with a plastic shaft and don gloves.

Locate the surface to swab. Choose a sump surface with low flow, the sump wall at the surface of the water, and remove any item preventing easy access to the surface. Next, unsheathe the swab by removing the outer packaging and expose the sterile cotton tip to the air.

Swab the sump wall over five to 10 centimeters to absorb the water and biofilm at the sump water surface level. Then sheathe the swab back or break the tip into a sterile centrifuge tube. Label the sample and send it for PCR testing or freeze at negative 80 degrees Celsius.

To perform sludge analysis by microscopy, use a 60 milliliter syringe to aspirate the sludge at the bottom of a sump. Divide the sample into 15 milliliter tubes. Screw on the caps and label the tubes.

Centrifuge the 15 milliliter tubes at 175 to 250 times the gravity for ten minutes in a centrifuge with swing buckets. Decant the tubes. After decanting, keep the sediments.

Next prepare the sugar saturated solution by mixing 227 grams of granulated sugar and 177 milliliters of hot water with a magnetic stirrer. Fill the tubes halfway with the sugar saturated solution. Screw on the caps and thoroughly mix the sediment with the solution.

Place the tubes in the centrifuge swing buckets and fill them up to the top with sugar saturated solution. Set one cover glass gently on top of each tube so it is contact with the sugar saturated solution. Note that the four tubes for each 60 milliliter sample are in case some cover glass falls and breaks during centrifugation.

Once the spin has completed, lift the cover glass and set it on a glass slide, then label the slide with a pencil or marker pen. In case too many cover glass breakages occur, fill up most of the tube with the sugar saturated solution and centrifuge the tube. Fill up to the top with the sugar saturated solution, then gently set the cover glass and wait for 30 minutes.

Look for the pseudocapillaria tomentosa eggs with the microscope. Identify the bipolar plugs at magnification 400X. To screen for imported animals in quarantine for P.tomentosa eggs, set male and female Danio rerio in a tank and add the in tank spawning tray but use it here to collect feces.

After one week remove the breeding device, and harvest the collected sludge in the breeding device as described previously. Aspirate the sludge at the bottom of a tank or sump with a 60 milliliter syringe and transfer the sample to a 60 milliliter tube. Close the tube with it's screw top and label the tube.

Dispose of the syringe. Shake the 60 milliliter tube and transfer 15 milliliters to a 15 milliliter tube. Close the tube with it's screw top and label the tube.

Centrifuge the 15 milliliter tube at 175 to 250 times the gravity for ten minutes in a centrifuge with swing buckets. Decant the tubes and keep the sediment in the tube. Unsheathe the swab by removing the outer packaging and expose the sterile cotton tip to the air.

Swab the sediment in the tube for 15 seconds. Sheathe the swab back or break the tip into a sterile centrifuge tube. Label the sample.

Freeze at negative 80 degree Celsius and send for PCR testing. Using the sump swab method of 115 fish tested, Mycobaterium chelonae was detected in five percent and Mycobaterium haemophilum was detected in three percent of the samples and were the only pathogenic species identified. From the same systems, 49 sump swabs revealed the presence of five Mycobaterial species.

Odds ratios show that the surface sump swab technique is a valuable alternative to the sole use of sentinels to screen the zebrafish facility for Mycobacterium. The environmental samples were also used to screen for Aeromonas hydrophila which were detected in fish, sludge and surface samples supporting the ability of the proposed techniques to screen the fish biotope. Mycobacterium chelonae and Mycobacterium fortuitum are detected more frequently by PCR in the surface sump swabs than in the fish sample.

Once mastered sampling takes only a few minutes and the detection of pseudocapillaria tomentosa eggs can be done in an hour. This technique makes environmental screening a key part of the routine health monitoring program. These methods can help answer key questions in the field of fish pathology, such as the relation between bacteria biofilm and bacterial infection.