Nathan Bay and Dr. Richard A. Heckmann, Zoology
Despite drastic control and containment measures, the incidence of whirling disease is on the rise in the West. Whirling disease occurs when the trophozoite stage of Myxobolus cerebralis invades the cartilage and organ of equilibrium of salmonids. Pathological symptoms include tail chasing behavior, blackened tafls, and vertebral abnormalities. Pan of the reason for the spread of the disease is the high occurrence of subclinical infections in fish that are transported into areas formerly free of the disease.
The viable spores remain in the fish cranium until the fish dies. Because the trophozoite invades cartilage, adult fish rarely are at risk of contracting the disease. Unfortunately, many fish, including adults, have subclinical infections that are difficult to detect, and of the disease. Diagnosis of this disease is economically important for fisheries.
Currently, diagnostic methods rely on clinical signs of the disease coupled with histological sampling. All current diagnostic techniques require destruction of the fish. Though fluorescent antibody techniques (FATs) have been attempted,1,2,3 it has been demonstrated that for both direct and indirect FATs extensive cross-reactivity with other myxosporeans, temperature effects, and age effects” pose major obstacles to diagnostic use of the FATs. 1
Initially, we hoped to use the Scanning Electron Microscope to better understand the morphology and fine structure of M. cerebralis. We fixed samples of cartilage from infected fish in 2% cacodylate buffered glutaraldehyde. After fixation, samples were post-fixed in 2%OsO4, dehydrated in a graded ethanol series, critical point dried, gold sputter coated, and mounted for SEM work. In the resulting micrographs, we were unable to isolate the organism. We hypothesized that the cartilage, though infected, did not contain any organism on the surface. We repeated the procedure with sectioned pieces of cartilage, and again did not find satisfactory results. We then attempted to use the pepsin digest method 4 for isolation of spores. We then prepared the samples using the OsO post- 4 fixation and graded ethanol series, this time air-drying droplets of the supernatant of the digest prior to sputtercoating. Again no samples of the parasite were viewed. Next, we took infected cartilage in distilled H20 and crushed it with a mortar and pestle, followed by the OsO4 post-fixation, dehydration, air-drying, and sputter coating. Though organisms were seen in the micrographs, they were not morphologically distinct due to what we assumed to be a proteinaceous coat covering the parasite. Methods of isolation of clean spores are still being attempted.
However, with the assistance of UVRMC Hematology Laboratory, we were able to analyze the blood chemistry of infected and non-infected trout (Oncorhynchus mykiss), verified by histological sampling. The results, which follow, revealed higher than normal initial levels of enzyme acid phosphatase and lower levels of blood Ca2+, and alkaline phosphatase in infected fish.
While not yet diagnostic, in the future samples of fish blood alone may be used to test for infections. Easy detection of sub-clinical infections will significantly reduce the spread of the parasite, helping to control the disease.
Because of the lower levels of blood Ca , enzyme acid phosphatase, 2+ and increased levels of alkaline phosphatase in the infected fish blood samples, we suspect an enzymatic breakdown of the cartilage. We are currently attempting to isolate an enzyme peptidase (couagenase or hyaluronidase) that breaks down the fish cartilage. This promises greater elucidation on the mechanism involved for cartilage damage, and the genetic basis for immunity in some fish species. It is hoped that in the future, isolation of the enzyme or enzymatic pathway will lead to better methods of control.
References
- Griffin, B. and E. Davis. 1978. Journal of Fish. Res. Board Can. 35: 1186-1190.
- Markiw, M. and K. Wolf 1978. Journal of Fish. Res. Board Can. 35: 828-832.
- Pauley, G. 1974. Journal of Fish. Res. Board Can. 31: 1481-1484.
- Thoesen, J. 1990. American Fisheries Society Blue Book: Suggested Procedures for the Detection and Identification of certain finfish and 4
shellfish pathogens.