Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

Jones, Simon R. M.; Goh, Benjamin; Prosperi-Porta, Gina

doi:10.1016/s0044-8486(02)00632-4

Cited by 14 publications

(17 citation statements)

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“…Previous ISH studies have examined the utility of different fixatives for detecting parasitic rDNA in infected fish tissues (Antonio et al 1998, Frasca et al 1999, Morris et al 1999, Sanchez et al 1999, Jones et al 2003. Our mRNA ISH test showed that fish tissues fixed for 48 h with Davidson's solution sufficiently inactivated RNases without damaging tissue morphology or probe hybridization.…”

Section: Discussionmentioning

confidence: 71%

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: 71%

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

et al. 2004

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“…Aliquots of 1.5 µl of extracted DNA (<50 µg ml -1 ) were then used as template in PCR reactions to amplify a 531 base-pair fragment of the Kudoa thyrsites 18S rRNA gene (Jones et al 2003).…”

Section: Methodsmentioning

confidence: 99%

“…Tissues from PCR-positive fish were assayed by ISH using the protocol of Jones et al (2003). Briefly, tissue sections were de-paraffinized in an increasing alcohol gradient and permeabilized for 10 min in cold (-20°C) acetone.…”

Section: Methodsmentioning

confidence: 99%

“…Mature spores and plasmodia of Kudoa thyrsites are detected by microscopic examination of fresh or histological preparations of skeletal muscle (Whitaker & Kent 1991), by PCR (Hervio et al 1997) or by in situ hybridization (ISH) (Jones et al 2003). Earlier developmental stages of the parasite are neither commonly reported nor well characterized.…”

Section: Introductionmentioning

confidence: 99%

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Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Young¹,

Jones²

2005

Dis. Aquat. Org.

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Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10°C (= 600 degree-days, °D). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 °D exposure, but they did react with ISH-identified stages following a 1600 °D exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 °D exposures, suggesting that the parasite observed in 600 °D infections represents an antigenically distinct developmental stage of K. thyrsites.

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“…Tissue sections mounted on AAS slides were deparaffinised in xylene and baked overnight at 60°C. The probe was used at a concentration of 0.3 ng/µl and incubated at 40 ng per section following the method of Jones et al (2003). Stained sections were examined with a compound microscope (400×).…”

Section: Digoxygenin (Dig)-labelled Probe and In Situ Hybridisation (mentioning

confidence: 99%

Proliferative renal myxosporidiosis in spawning coho salmon (Oncorhynchus kisutch) in British Columbia and Washington

Jones¹,

Prosperi-Porta²,

Dawe³

et al. 2004

FOLIA PARASIT

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Abstract. An unidentified myxosporean parasite (CKX) is described from the kidney of approximately 80% of spawning coho salmon Oncorhynchus kisutch (Walbaum) in British Columbia, Canada and Washington, United States of America. Morphological features were described using light and electron microscopy. Sequencing of polymerase chain reaction (PCR) amplified 18S ribosomal RNA gene and in situ hybridisation were used to further characterise CKX. The parasite occurred with a focal distribution within tubule epithelial cells, the tubule lumen and the interstitium as primary cells containing from one to at least 16 secondary cells. Luminal stages were degenerate and sporogony was not observed. In situ hybridisation using a digoxygenin-labelled DNA probe confirmed CKX to be the source of DNA used in PCR analyses. CKX 18S rDNA sequences were most similar (97%) to those of Sphaerospora oncorhynchi. Phylogenetic analysis revealed similarities among the 18S rDNA sequences of CKX, S. oncorhynchi and Myxidium lieberkuehni. CKX is hypothesised to be the abortive extrasporogonic development of a Sphaerospora sp. or Myxidium sp. occurring in immune-incompetent spawning and senescent salmon.Elevated pre-spawn mortality was observed among returning stocks of adult coho salmon Oncorhynchus kisutch (Walbaum) migrating in streams along northern Vancouver Island. Analysis of specimens from the initial diagnostic case indicated that the furunculosis bacterium Aeromonas salmonicida contributed to the mortality. Routine histological examination of kidney from moribund and fresh-dead salmon also revealed foci of infection with intracellular parasites that resembled extrasporogonic myxosporean stages. This pattern of infection had not previously been noted in coho salmon. The present study describes the parasite in coho salmon, herein referred to as the unknown coho kidney parasite (CKX), and reports on its occurrence in Canada and the United States. MATERIALS AND METHODSCoho salmon locations and samples. Samples of posterior kidney, liver, spleen and gill were collected from fresh spawned salmon belonging to Marble, Cluxewe, Washlawis, Waukwaas, Quatse and Stephens Rivers stocks in northern Vancouver Island, British Columbia, Canada. All dissecting tools were carefully cleaned between fish by rinsing first with water, then bleach, then water, then 95% ethanol followed by flaming. From each organ sample, small (1-2 mm 3 ) pieces were preserved in 2% glutaraldehyde in 0.1 M Sörensen's phosphate buffer (pH 7.2) and two larger (10-20 mm 3 ) pieces were fixed in 95% ethanol and Davidson's solution, respectively. Similarly, duplicated 10-20 mm 3 kidney samples collected from the Quinault, Sooes, Methow and Wenatchee Rivers, Washington, United States of America were fixed in Davidson's solution and 95% ethanol, respectively.Histological analysis. Tissues preserved in Davidson's solution were dehydrated through isopropanol, cleared in xylene and embedded in paraffin wax. Sections (5 µm) were mounted on glass slides and stained with haematoxylin ...

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Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

Cited by 14 publications

References 17 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

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Proliferative renal myxosporidiosis in spawning coho salmon (Oncorhynchus kisutch) in British Columbia and Washington

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