Evaluation of tissue changes following intramuscular infiltration of lidocaine in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss were infiltrated with either saline or lidocaine adjacent to the dorsal fin to assess histopathological changes. Infiltration was done as if it were being used as a local anaesthetic. Tissue lesions and associated tissue healing were examined over a period of 30 days. Most changes occurred at the cranial site of where the solution was first infiltrated. The infiltration of a dose of 10 mg kg-1 of lidocaine appears to have damaged the skeletal muscle and connective tissues more than a similar volume of saline, especially during the first 15 days. The primary changes included haemorrhage, inflammation and muscle degeneration and necrosis. By day 30 post-infiltration inflammatory lesions were either nearly or completely absent, signs of myofibre regeneration were noted in only one fish. This experiment shows local anaesthetics and saline can produce localized tissue damage, especially during the first 2 weeks post infiltration. Care should be taken to allow the fish to heal for at least 30 days and probably more, no matter the solution administered, especially if giving repeated injections or infiltrations at the same site.

CpG inclusion in feed reduces sea lice, Lepeophtheirus salmonis, numbers following re-infection.

Lepeophtheirus salmonis infections in Atlantic salmon, Salmo salar, have been characterized by little to no hyperplastic response and a biphasic immune response that results in chronic inflammation with tissue repair as the infection progresses. We hypothesized that CpG administration with prior lice exposure would enhance epithelial inflammatory mechanisms and boost the Atlantic salmon immune response to L. salmonis, leading to greater protection against infection. We administered multiple exposures of L. salmonis to two groups of Atlantic salmon and compared responses against first-time exposed Atlantic salmon. Following re-exposure, CpG fed fish exhibited increased skin expression of interleukin (IL)-1ß and IL-12 ß compared to control previously exposed (CPE) and control first-time exposed (CFE) animals, respectively. This inflammatory enhancement occurred with significantly lower expression of matrix metalloproteinase-9 (MMP 9), both systemically (spleen) and locally (skin). Reduced MMP 9 expression was a hallmark of the re-infected fish (occurred in both tissues at both times). When significant differences were present in the skin or spleen, the two re-exposed groups showed greater similarity than with the first exposure group. Lice numbers on CpG fed fish were significantly lower than CFE fish at 7 days post-re-infection (dpri), and although they were not significantly different at 17 dpri, the trend of lower lice levels remained. CpG fed fish also showed nearly twofold greater protection than CPE when compared to the CFE group (48.5% vs. 27.0% reductions at 7 dpri and 27.2% vs. 13.1% reductions at 17 dpri, respectively). The enhanced protection of CpG oligodeoxynucleotide administration to previous exposure was consistent across all body surfaces and suggests that CpG can not only enhance innate responses to L. salmonis in Atlantic salmon, but also further stimulate adaptive responses.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

Pathology associated with an aquareovirus in captive juvenile Atlantic halibut Hippoglossus hippoglossus and an experimental treatment strategy for a concurrent bacterial infection.

A large-scale mortality of larval and juvenile halibut Hippoglossus hippoglossus occurred at a semi-commercial halibut farm in Atlantic Canada. Investigation of the cause revealed aquareovirus particles in necrotic liver tissue of affected fish. Cytopathic effect on CHSE-214 cell lines occurred from all fish cultured for viruses, and the viral morphology of the particles in culture was consistent with that observed in necrotic host tissue. The virus was placed in the family of Reoviridae, genus Aquareovirus based on morphology and RT-PCR results. Multifocal hepatocellular necrosis was a consistent finding in all fish as well as acute necrosis of proximal renal tubules. Concurrent bacterial infections were present in some specimens. Fish experimentally treated with oxytetracycline or a combination of oxytetracycline and chloramine-T had a significantly lower mortality rate than untreated fish. Fish treated with chloramine-T alone had a significantly elevated mortality rate compared to controls. Despite supportive medical therapy, mortality levels in treated and untreated groups remained elevated, supporting the hypothesis that the primary pathogen was of viral origin. This is the first report of elevated mortalities in Atlantic halibut associated with an aquareovirus.

Virulence and antigenic characteristics of a cultured Rickettsiales-like organism isolated from farmed Atlantic salmon Salmo salar in eastern Canada.

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.