Comparison of single-round polymerase chain reaction (PCR) and pepsin-trypsin digest (PTD) methods for detection of Myxobolus cerebralis

Schisler, George J.; Bergersen, Eric P.; Walker, Peter G.; Wood, John S.; Epp, Janet K.

doi:10.3354/dao045109

Cited by 25 publications

(42 citation statements)

References 14 publications

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“…Prior studies (Andree et al 1998, Antonio et al 1998, Baldwin & Myklebust 2002, Schisler et al 2001) have reported effective molecular methods for detecting Myxobolus cerebralis, but these tests provided no biological explanation for parasite activity during disease development. Using MyxSP-1 as the target gene, our real-time TaqMan PCR test detected protease transcription from early, intermediate, and late developmental stages of the parasite (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2004

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Serine proteases have been recognized as key factors in parasite physiology and disease development. We have identified a serine protease gene from Myxobolus cerebralis, MyxSP-1, the myxozoan parasite causing whirling disease in salmonid fishes. The amino acid sequence, as deduced from the cDNA sequence, included a catalytic residue arrangement similar to that of the chymotrypsin family of serine proteases. A real-time TaqMan polymerase chain reaction (PCR) analysis revealed differences in the transcription levels for the chymotrypsin-like protease as found in early, intermediate, and late developmental stages of the parasite in experimentally-infected rainbow trout Oncorhynchus mykiss. MyxSP-1 transcription differed between individual tissues at each sampling point and in the same tissues over time (p < 0.0001). A nonradioactive mRNA in situ hybridization (ISH) protocol was developed to detect MyxSP-1 transcripts. Using a mixture of 3 digoxigenin-labeled antisense mRNA probes, MyxSP-1 transcription was observed in developmental stages of the parasite during the acute and chronic phases of the disease over a 240 d time period in infected rainbow trout tissues. MyxSP-1 transcription observed by ISH in cartilage and as associated with cartilage destruction was consistent with our real-time TaqMan PCR findings that demonstrated high levels of MyxSP-1 transcription during lesion development. Identifying genes encoding these enzymes and characterization of their functions can lead to the development of new chemotherapeutic protocols and vaccine approaches to control parasitic diseases.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2004

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“…Ten fish were sampled prior to exposure and individually tested for the presence of nucleic acid of the Myxobolus cerebralis parasite by a polymerase chain reaction (PCR) test (Andree et al 1998), modified as a single round procedure (Schisler et al 2001).…”

Section: Methodsmentioning

confidence: 99%

Response of rainbow trout Oncorhynchus mykiss to exposure to Myxobolus cerebralis above and below a point source of infectivity in the upper Colorado River

Thompson¹,

Nehring²,

Bowden³

et al. 2002

Dis. Aquat. Org.

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We exposed 9 wk old rainbow trout Oncorhynchus mykiss to ambient levels of Myxobolus cerebralis infectious stages at 4 sites of suspected differing infectivity in the Colorado River. Exposure was estimated by periodic filtration of river water at each exposure location. After a 32 d exposure, the fish were held in the Colorado River at a common site for over a year. Resulting infection was evaluated by the presence of clinical signs (whirling behavior, cranial deformity/exophthalmia, and black tail), severity of microscopic lesions, and myxospore counts (8, 10, 12, and 14 mo post-exposure). Two exposure sites that were immediately downstream of Windy Gap Reservoir were much higher in infectivity than the site above the reservoir or the site 26 km downstream of the reservoir. Rainbow trout exposed at those locations showed higher prevalence of clinical signs of whirling disease, more severe histological evidence of infection and higher average myxospore concentrations than those exposed above the reservoir or 26 km below the reservoir. Many more M. cerebralis actinospores were observed from water filtration at the 2 sites immediately below the reservoir compared to the other sites. KEY WORDS: Myxobolus cerebralis · Whirling disease · Rainbow trout Resale or republication not permitted without written consent of the publisherDis Aquat Org 49: [171][172][173][174][175][176][177][178] 2002 Schisler et al. concluded that their results 'tend to support the hypothesis that whirling disease is the predominant cause of declines in fingerling rainbow trout survival in some Colorado rivers, with other stressors playing an important but secondary role. ' By early 1997, more prevalent and severe clinical signs of whirling disease among young-of-the-year brown trout Salmo trutta and rainbow trout from the Colorado River in the proximity of Windy Gap Reservoir indicated that infection rates were higher below the reservoir than in areas further downstream (Nehring 1998). This suggested that the area near the reservoir was a point source of infectivity, but whether the source was the reservoir or the spill basin was unclear. Trout migrating upstream would be stopped in the spill basin below the dam, and we hypothesized that many might remain there only to be stranded and perhaps die in the spill basin when releases ceased for the winter (winter water releases occur through a separate channel). These fish, if infected, could then be a source of Myxobolus cerebralis infection in the Tubifex tubifex population.These observations were the impetus for an experiment conducted in 1997 to 1998. We conducted a sentinel fish test designed to expose the fish to differing levels of Myxobolus cerebralis infectious units, and determine if the spill basin or Windy Gap Reservoir was the primary source of infectivity in this area. MATERIALS AND METHODSWe chose 4 exposure locations in the Colorado River that we believed represented differing levels of infectivity (Fig. 1). The uppermost location was 0.5 km upstream of Windy Gap Res...

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“…1986) and testing each individual T. tubifex was cost prohibitive. These composite samples were analyzed by a single-round polymerase chain reaction (PCR) assay described in Schisler et al (2001) to determine the presence of M. cerebralis DNA, using a modification of the technique described by Andree et al (1998).The proportion of infected Tubifex tubifex was calculated using composite sampling statistics (Zendt & Bergersen 2000). Infection rates were calculated using the equation given by Boswell & Patil (1987): where is the unknown prevalence of infection, n is the number of individuals per composite sample, m is the number of composite samples, and p m is the proportion of the composite samples that were positive for Myxobolus cerebralis DNA.…”

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confidence: 99%

Factors influencing the distribution of Myxobolus cerebralis, the causative agent of whirling disease, in the Cache la Poudre River, Colorado

Allen

Bergersen

2002

Dis. Aquat. Org.

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Oligochaetes, triactinomyxons (TAMs), and age-0 trout were sampled in the upper Cache la Poudre River, Colorado, to determine the distribution of Myxobolus cerebralis during 1997 and 1998. Densities of the intermediate host, the oligochaete Tubifex tubifex, were 3.5 orders of magnitude higher in the M. cerebralis-infected Poudre Rearing Unit (PRU) trout rearing ponds than at any of the river sampling reaches. Oligochaetes, including T. tubifex, were rare in the river (1 oligochaete m -2 ), except in a few stream side alcoves and eddies (50 oligochaete m -2 ). Species composition of oligochaetes in the river reaches was more diverse than in the PRU. Tubifex tubifex constituted 50% or less of the oligochaete community in the river and 98% in the PRU. Infection rates of T. tubifex were 1% in the area above the PRU, 2% in the PRU, and 6% below the PRU. An increased M. cerebralis intensity of infection in age-0 trout below the PRU could not be attributed entirely to the high numbers of TAMs in its effluent (3.7 TAMs l -1 ). Low densities of TAMs ranging from 0 to 0.2 TAMs l -1 were found in the river reaches, yet nearly all of the age-0 trout were infected soon after emergence. This suggests that very few TAMs, as measured by filtration, need be present in the water column to bring about infection in the majority of trout present. This also indicates that the parasite can persist and potentially cause reduced juvenile trout recruitment in cold, oligotrophic, sediment poor, highgradient streams. KEY WORDS: Salmonid whirling disease · Myxobolus cerebralis · Tubifex tubifex · Trout rearing unit Resale or republication not permitted without written consent of the publisherDis Aquat Org 49: [51][52][53][54][55][56][57][58][59][60] 2002 Wildlife, pers. comm.). Rainbow trout populations in at least 370 km of stream have been affected .Myxobolus cerebralis was first described in Germany in 1893 (Hoffman 1990). Though it is believed to be endemic to Europe , it was not detected until rainbow trout, imported from the USA to a German trout rearing facility, displayed classic WD signs (Hoffman 1990). The mechanism of infection remained unknown for almost a century until Markiw & Wolf (1983) found that tubificid worms were a necessary alternate host for the parasite. Antigenic and genetic evidence has since confirmed the relationship between triactinomyxons (TAMs), which are the waterborne, fish-infective stage of the parasite, and the myxospore, which infects Tubifex tubifex (Markiw 1989, Andree et al. 1997. The oligochaete host for M. cerebralis is apparently restricted to T. tubifex; attempts to infect other oligochaete species with M. cerebralis myxospores have been unsuccessful (Wolf et al. 1986, El-Matbouli & Hoffman 1989, Hedrick et al. 1996.Myxobolus cerebralis was first detected in the Cache la Poudre River in 1988 (Nehring 2000). Drastic declines in the number of age-1 wild rainbow trout became evident in the early 1990s at 4 different monitoring stations in a 30 km reach of the river. In 1995, 5 yr after ...

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Comparison of single-round polymerase chain reaction (PCR) and pepsin-trypsin digest (PTD) methods for detection of Myxobolus cerebralis

Cited by 25 publications

References 14 publications

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

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

Response of rainbow trout Oncorhynchus mykiss to exposure to Myxobolus cerebralis above and below a point source of infectivity in the upper Colorado River

Factors influencing the distribution of Myxobolus cerebralis, the causative agent of whirling disease, in the Cache la Poudre River, Colorado

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