Pepsin-Trypsin Digestion (PTD) for Myxobolus cerebralis testing

Certification testing to screen fish for M. cerebralis before export or stocking almost always requires following methodology from the American Fisheries Society-Fish Health Section Blue Book’s USFWS/AFS-FHS Standard Procedures for Aquatic Animal Health Inspections. This test involves performing a pepsin-trypsin digest on cartilage from fresh or frozen samples and then visualizing the spores under a microscope. If myxospores consistent with M. cerebralis are visualized, PCR and histopathology testing can be performed as confirmatory testing.

Samples are heated until flesh is soft and eyes are opaque. Then all flesh is removed from the samples and cartilage, bone, gill arches, and opercles are saved for testing.

Specimens and beakers on countertop in lab.
Counts are verified prior to testing to confirm actual numbers of specimens submitted match what is listed on the accession form.
Up to five individual fish can be pooled for testing. Heads are cut in half and one half is retained for confirmatory if necessary.
Up to five individual fish can be pooled for testing. Heads are cut in half and one half is retained for confirmatory if necessary.
Samples being heated prior to defleshing.
Samples being heated prior to defleshing.
Pepsin-Trypsin Digestion (PTD) for Myxobolus cerebralis testing Certification testing to screen fish for M. cerebralis before export or stocking almost always requires following methodology from the American Fisheries Society-Fish Health Section Blue Book’s USFWS/AFS-FHS Standard Procedures for Aquatic Animal Health Inspections. This test involves performing a pepsin-trypsin digest on cartilage from fresh or frozen samples and then visualizing the spores under a microscope. If myxospores consistent with M. cerebralis are visualized, PCR and histopathology testing can be performed as confirmatory testing. Defleshing: Samples are heated until flesh is soft and eyes are opaque. Then all flesh is removed from the samples and cartilage, bone, gill arches, and opercles are saved for testing. Counts are verified prior to testing to confirm actual numbers of specimens submitted match what is listed on the accession form. Up to five individual fish can be pooled for testing. Heads are cut in half and one half is retained for confirmatory if necessary. A pool of heads in a beaker ready to be heated up prior to defleshing. Samples being heated prior to defleshing. A head ready for defleshing. Pepsin and Trypsin Digestion: The bone, cartilage, gill arches, and opercles are digested using pepsin and trypsin to small particles to release spores. Beakers of trypsin ready to be decanted for the concentration step. Digest product being prepared for the concentration step. Dextrose Centrifugation: This step concentrates spores released from the digest into a pellet. Digest product being poured into tubes prior to centrifugation. Final digest product balanced for centrifugation. This step concentrates the spores in a pellet that is used to prepare slides for microscopic evaluation. Analyzing Samples: Slides are prepared with pelleted digest material and evaluated microscopically for myxospores. Slides being prepared for staining and subsequent microscopic evaluation.
A pool of heads in a beaker ready to be heated up prior to defleshing.
Lab technician point at specimen.
A head ready for defleshing.

The bone, cartilage, gill arches, and opercles are digested using pepsin and trypsin to small particles to release spores.

Countertop in a WADDL lab.
Beakers of trypsin ready to be decanted for the concentration step.
Lab technician wearing gloves.
Digest product being poured into tubes prior to centrifugation.
Countertop in a WADDL lab with equipment on it.
Digest product being prepared for the concentration step.
Photo of centrifuge.
Final digest product balanced for centrifugation. This step concentrates the spores in a pellet that is used to prepare slides for microscopic evaluation.

Slides are prepared with pelleted digest material and evaluated microscopically for myxospores.

Slides being prepared for staining and subsequent microscopic evaluation.
Slides being prepared for staining and subsequent microscopic evaluation.

Slides being prepared for staining and subsequent microscopic evaluation.