A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

Abstract

A nested polymerase chain reaction (PCR) test was developed to amplify a segment of the 18s rRNA gene from Myxobolus cerebralis, the agent causing whirling disease in salmonid fish. The PCR amplifies a 415 bp amplicon that was identified by dideoxynucleotide terminated sequencing to be identical to the known 18s rDNA sequence of M. cerebralis. There was no amplification of genomic DNA from 4 other myxosporean parasites of salmonid fish from the genus Myxobolus incl.uding M. arcticus, M. insidiosus, M, neurobius, and )\/I. squamalis. The efficacy of the PCR test to detect early infections was demonstrated by amplificat~on of the 415 bp fragment from experimentally exposed rainbow trout Oncorhynchus mykiss at 2 h and at 1, 2, and 3 wk postexposure to actinosporean stages (triactinomyxons) of M. cerebralis. In contrast, standard microscopic examinations of stained tissue sections of the same fish used for PCR were less reliable in detecting the presence of the parasite Additional examinations of fish 5 mo postexposure, after sporogenesis had occurred, found the PCR to be a more reliable indicator of infection than the pepsin-trypsin digest (PTD) method, paricularly when trout were experimentally exposed to low levels of the infectious stages of the parasite. The PCR was able to amplify to detectable levels the equivalent of a single sporoplasm of M. cerebralis as found in a tissue sample. This test improves the detection of M. cerebralis because it can detect the presence of the parasite: (1) in both hosts, (2) in all known stages of its life cycle, and (3) at lower thresholds than currently used diagnostic methods. Lastly, the PCR test I S less susceptible to morphological mlsidentifications of the spores that can occur with current microscopic procedures.

A Review: Gaps in Our Knowledge on Myxozoan Parasites of Fishes

“…One explana tion of this inconsistent production of antibodies is that myxozoans may mimic antigens of host fish (Pauley, 1974). Acquired resistance to reinfection with PKD and whirling disease has been documented (Klontz et al, 1986;Foott and Hedrick, 1987;Hedrick et al, 1998) Introduction of the resistant strains The genetic resistance of certain salmonid fish spe cies or strains to C. shasta and M. cerebralis have been reported (Bartholomew, 1998;Hedrick et al, 1999aHedrick et al, , 1999Thompson et al, 1999). However, salmonid strains resistant to C. shasta were found to be suscep tible to M. cerebralis, suggesting that the mechanisms of resistance are different for the two myxozoans (Hedrick et al, 2001).…”

“…However, a threat of parasite transmission via fish eggs is unlikely (Markiw, 1991). To enforce the quarantine policy rigorously, molecular-based detection methods will be useful for sensitive diagnosis of parasites even from live and asymptomatic fish (Andree et al, 1998;Fox et al, 2000). Selection of disease-free sites (avoidance of infected waters) is worthy of consideration, as some myxozoans have only a limited geographical distribution (Hendrickson et al, 1989;Sugiyama et al, 1999).…”

“…For M. cerebralis, it has been well documented that delaying exposure of rainbow trout to at least 9 weeks post hatch greatly decreases the incidence of whirling disease Biological control Some species or strains of oligochaetes, resistant to Myxobolus cerebralis, have been shown to be capable of ingesting and inactivating the myxospores of M. cerebralis (Hedrick et al, 1998;El-Matbouli et al, 1999*: see the footnote of the previous page). Benthos-eating fishes may be useful for reducing the biomass of anne lids in pond bottoms .…”

Polymerase Chain Reaction and Indirect Fluorescent Antibody Technique for the Detection of Kudoa amamiensis (Multivalvulida: Myxozoa) in Yellowtail Seriola quinqueradiata.

“…Andree et al (1998) indicated that the nested PCR could detect even a single cell of the actinospore stage of Myxobolus cerebralis. Saulnier and de Kinkelin (1997) reported that as few as 5 PKX cells were enough for detection by the PCR assay.…”

“…The need for accurate identification of the parasite in all developmen tal stages has led many researchers to pursue new molecular and immunological approaches for detection of several myxosporeans (Markiw and Wolf, 1978;Bartholomew et al, 1989Bartholomew et al, , 1995Saulnier and de Kinkekin, 1997;Andree et al, 1998;Palenzuela et al, 1999). In the present study, therefore, we aimed to develop highly sensitive and specific detection methods for K. amamiensis, and to follow the development of the parasite in the musculature of yellowtail.…”