Myxosoma cerebralis: Isolation and Concentration from Fish Skeletal Elements — Sequential Enzymatic Digestions and Purification by Differential Centrifugation

Abstract

An effective sequential procedure for recovery of Myxosoma cerebralis spores from infected trout was developed, and quantification of spores was carried out at each step of release and concentration. Methods are described for fresh and frozen material. Effective concentration of from 1100- to 9000-fold and an estimated efficiency of recovery of about 80% has been achieved. Tabular and graphic data are presented with recommendations for diagnostic applications. The immediate applications of these procedures are in implementing more effective detection and in preparing antigen for the immunologic studies that should provide the most sensitive detection.

Identification of a serine protease gene expressed by Myxobolus cerebralis during development in rainbow trout Oncorhynchus mykiss

“…A population of known susceptible aquatic oligochaetes was maintained in aquaria for the production of the triactinomyxon stage of Myxobolus cerebralis. The oligochaetes were exposed to myxospores freshly isolated (Markiw & Wolf 1974) from experimentally infected rainbow trout as previously described (Andree et al 1998). Beginning at 90 d post-exposure, triactinomyxons released in the aquarium water were harvested and counted (Markiw 1989, Andree et al 1998.…”

“…Detecting M. cerebralis among populations of captive or wild fish stocks is a key step in controlling the spread of whirling disease. Currently, a presumptive diagnosis of whirling disease is dependent on morphological identification of myxospores released by mechanical and chemical treatments from skeletal tissues of infected trout or salmon (Markiw & Wolf 1974, O'Grodnick 1975. Detection of spores or presporogonic stages in cartilage in hematoxylin and eosin (H&E)-stained tissue sections have been used as a confirmatory test for M. cerebralis (Hedrick et al 1999a, Baldwin et al 2000.…”

Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

“…The principal methods used to identify the parasite include pepsintrypsin digest (PTD) (Markiw & Wolf 1974), histopathology (Lorz & Amandi 1994, Hedrick et al 1999, Baldwin et al 2000 as modified by Andree et al 2002) and polymerase chain reaction (PCR) analysis (Andree et al 1998, Schisler et al 2001, Baldwin & Myklebust 2002. Histopathology and PTD analysis can also be used to determine infection severity, but both techniques lack sensitivity for detecting low-level infections.…”

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease

“…As described previously, individual heads were used both for histological analyses and spore detection and enumeration. One half of the head was subjected to pepsin trypsin digestion as described by Markiw & Wolf (1974a) with minor modifications. Fish were scored as infected if any spores were observed.…”