In situ hybridization of DNA probes to PKX, the causative organism of proliferative kidney disease (PKD)

Proliferative kidney disease (PKD) is a commercially important disease of salmonids in both North America and western Europe.Little is known of the life-cycle of the causative organism referred to as PKX.The PKX parasite appears in the kidney interstitium of fish 2±4 weeks after exposure to enzootic water.In the kidney, the parasite rapidly proliferates, becoming the focus of an intense inflammatory response (Kent & Hedrick 1986;Clifton-Hadley, Bucke & Richards 1987).Twelve weeks post-exposure, the parasites start to display degenerative changes, and after 20 weeks, many fish have made a complete recovery from the disease.The PKX parasite has two developmental phases in the kidney, referred to as the extrasporogonic and sporogonic stages.The extrasporogonic stages occur in the kidney interstitium, and are composed of a primary cell containing one or more secondary cells.The secondary cells are released from the primary cell to form the sporogonic stages of the parasite in the kidney tubule lumen (Marin de Mateo, Adams, Richards, Castagnaro & Hedrick 1993; Morris, Adams & Richards 1997).Monoclonal antibodies have been developed against PKX and have demonstrated that the antigenic characteristics of the parasite change during its development in the fish (Adams, Richards & Marin de Mateo 1992; Marin de Mateo, McGeorge, Morris & Kent 1996; Saulnier & de Kinkelin 1996;Morris et al. 1997).This change in antigenicity suggests that monoclonal antibodies are limited for studies into the life-cycle of PKX and for detecting early parasite infections.Recently, primers which hybridize to the DNA of PKX have been designed (Saulnier, Bremont & de Kinkelin 1996;Saulnier & de Kinkelin 1997; Kent, Khattra, Hervio & Devlin 1998).These primers will be invaluable for the study of the parasites' life-cycle.To aid in this work, an in situ hybridization technique has been developed to detect PKX.Kidneys from rainbow trout which were positive for PKX were fixed in 10% buffered formalin and embedded in paraffin wax.Sections were cut at 7 mm and adhered to aminoalkylsilane-treated glass slides.The sections were dewaxed through xylene (2 ´5 min), 100% alcohol (5 min) and 70% alcohol (3 min), and then rehydrated in distilled water.To permeabilize the tissue, the sections were exposed to 0.2 m hydrochloric acid for 20 min.Following a rinse in 2 ´standard saline citrate (SSC, 0.3 m tri-sodium citrate, 3 m NaCl, ´20), the sections were incubated with 0.1% Triton X 100 in phosphate-buffered saline (PBS, 0.02 m phosphate, 1.5 m NaCl, pH 7.2) for 5 min.After washing in tris-buffered saline (TBS, 0.05 m Tris, 0.15 m NaCl, pH 7.6), the slides were incubated at 37 °C for 20 min with 100 mg mL ±1 Proteinase K (Boehringer Mannheim GmBH, Mannheim, Germany) in TBS, pH 8.0.The slides were rinsed twice in 2% glycine/PBS for 3 min per rinse, followed by PBS.The sections were post-fixed in cold (4 °C) 0.4% paraformaldehyde in PBS for 20 min.Four DNA oligonucleotides, 3F, 4R, 5F and 6R,