title: Virulence and antigenic characteristics of a cultured Rickettsiales-like organism isolated from farmed Atlantic salmon Salmo salar in eastern Canada.
abstract: The present study describes culture, virulence and antigenic characteristics of a Rickettsiales-like organism (RLO) associated with mortality in farmed Atlantic salmon in eastern Canada. Clinical disease was reproduced in naive Atlantic salmon parr by intraperitoneal i.p. inoculation with kidney homogenate from naturally infected fish. Pure cultures of RLO were isolated into chinook salmon embryo (CHSE) cells from kidney of experimentally infected fish. The RLO caused cytopathic effect in cultured CHSE-214 typified by coalescing areas of swollen cells that eventually detached from the substrate. Bacteria in infected culture supernatants reacted with Piscirickettsia salmonis-specific polyclonal sera or monoclonal antibody (MAb) in an indirect fluorescent antibody test. IP inoculation with cultured RLO resulted in mortalities of 100, 62, 22.5 and 0% in Atlantic salmon, coho salmon, rainbow trout and common carp, respectively. Cultured RLO were sensitive to chloramphenicol, flumequine, oxytetracycline and oxolinic acid and insensitive to gentamicin and amphotericin B. RLO antigens were compared with those of 3 strains of P. salmonis from Chilean salmon by SDS-PAGE and immunoblotting. A silver-staining band of about 12 kDa was detected in proteinase K (PK) digests of all RLO strains, and a diffuse band of about 15 kDa was observed in 2 Chilean strains only. No other silver-stained bands were visible in PK digests of any strain examined. The polyclonal serum recognized 9 protein bands and multiple non-protein bands extending from less than 20 kDa to greater than 95 kDa in all isolates. The MAb reacted with an epitope in PK digests that occurred in all 4 strains on structures of widely ranging molecular masses, resulting in a ladder pattern similar to that obtained with polyclonal serum. Treatment of PK digests with periodic acid abolished reactivity with MAb and polyclonal serum. Co-elution of 2-keto-3-deoxyoctonate and MAb reactivity following size exclusion chromatography of solubilized P. salmonis suggested that the MAb recognized a lipopolysaccharide-associated epitope in all 4 RLO isolates. Cultural, virulence and antigenic similarities among the strains examined in the present study indicate that the eastern Canadian salmonid RLO should be considered a strain of P. salmonis.
authors.affiliation: Aqua Health Ltd, West Royalty Industrial Park, Charlottetown, Prince Edward Island, Canada. simjones@upei.ca
authors.forename: S R
authors.initials: SR
authors.lastname: Jones
authors.forename: R J
authors.initials: RJ
authors.lastname: Markham
authors.forename: D B
authors.initials: DB
authors.lastname: Groman
authors.forename: R R
authors.initials: RR
authors.lastname: Cusack
mesh_terms: D020561:Alphaproteobacteria / Q000145:classification / Q000276:immunology / Q000472:pathogenicity*; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D002170:Canada / Q000453:epidemiology; D002460:Cell Line; D005260:Female; D005393:Fish Diseases / Q000453:epidemiology / Q000382:microbiology*; D005398:Fisheries; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D016905:Gram-Negative Bacterial Infections / Q000453:epidemiology / Q000382:microbiology / Q000662:veterinary*; D015151:Immunoblotting / Q000662:veterinary; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D012474:Salmon; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial