Development of a Piscirickettsia salmonis immersion challenge model to investigate the comparative susceptibility of three salmon species.

Piscirickettsia salmonis, the aetiological agent of salmonid rickettsial septicaemia (SRS), is a global pathogen of wild and cultured marine salmonids. Here, we describe the development and application of a reproducible, standardized immersion challenge model to induce clinical SRS in juvenile pink (Oncorhynchus gorbuscha), Atlantic (Salmo salar) and sockeye salmon (O. nerka). Following a 1-hr immersion in 105 colony-forming units/ml, cumulative mortality in Atlantic salmon was 63.2% while mortality in sockeye salmon was 10%. Prevalence and levels of the bacterium in kidney prior to onset of mortality were lower in sockeye compared with Atlantic or pink salmon. The timing and magnitude of bacterial shedding were estimated from water samples collected during the exposure trials. Shedding was estimated to be 82-fold higher in Atlantic salmon as compared to sockeye salmon and peaked in the Atlantic salmon trial at 36 d post-immersion. These data suggest sockeye salmon are less susceptible to P. salmonis than Atlantic or pink salmon. Finally, skin lesions were observed on infected fish during all trials, often in the absence of detectable infection in kidney. As a result, we hypothesize that skin is the primary point of entry for P. salmonis during the immersion challenge.

Caligus rogercresseyi infestation is associated with Piscirickettsia salmonis-attributed mortalities in farmed salmonids in Chile.

Caligidosis and Piscirickettsiosis are currently the most important sanitary challenges for the Chilean salmon industry. Caligidosis is caused by the ectoparasite, Caligus rogercresseyi and Piscirickettsiosis is caused by the intracellular bacterium, Piscirickettsia salmonis. Both diseases are highly prevalent and widely distributed in farming areas in Chile. The co-occurrence of the two diseases is frequently reported on salmon farms. However, there is little epidemiological evidence as to whether these two diseases are associated and generate interactive effects. This study was undertaken to evaluate the potential effects of C. rogercresseyi infestation on P. salmonis-attributed mortalities in farmed salmonids in Chile. Using a linear regression model, the potential association between the mean abundance of adult C. rogercresseyi in a period of 10 weeks and Piscirickettsiosis cumulative mortalities observed in the following 10 weeks was evaluated, while controlling for important confounders. These two 10-week windows were set around the time-point at which Piscirickettsiosis weekly mortality exceeded 0.1% for the first time in a production cycle. We found that the mean abundance of adult C. rogercresseyi was significantly associated with the Piscirickettsiosis cumulative mortality, suggesting the two diseases have a synergistic relationship. This relationship was of the same intensity in Atlantic salmon and rainbow trout. Our findings highlight the importance of taking effective control measures for C. rogercresseyi as a part of the strategies in place to reduce P. salmonis-attributed mortalities on salmon farms in Chile.

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90-like isolate: A comparative study.

Piscirickettsiosis, caused by the intracellular Gram-negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90-like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90-like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real-time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.

A piscirickettsiosis-like syndrome in cultured Nile tilapia in Latin America with Francisella spp. as the pathogenic agent.

In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan).

Persistence of Piscirickettsia salmonis and detection of serum antibodies to the bacterium in white seabass Atractoscion nobilis following experimental exposure.

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis.

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.

Experimental infection of coho salmon Oncorhynchus kisutch by exposure of skin, gills and intestine with Piscirickettsia salmonis.

Piscirickettsiosis pathogenesis was examined using some tissues as entry portals of Piscirickettsia salmonis in coho salmon. Juvenile fish, weighing approximately 8.4 g, were used in this trial. Inocula were prepared using the strain SLGO-95 of P. salmonis. The micro-organism was cultured in the CHSE-214 cell line as described by Fryer et al. (1990) and doses containing 10(4.7) and 10(3.7) TCID50 were prepared. Each dose was used to infect the fish via skin, gills and intestine. Skin and gills were exposed by calibrated drops, and the intestine by an intubation through the anal opening. Some fish were injected intraperitoneally with the same P. salmonis doses, as positive virulence controls. Sham-inoculated fish for each of the tested routes were also included as negative controls. Piscirickettsiosis was experimentally reproduced with all the inoculation methods. Cumulative mortalities and survival analyses showed that the most effective entry portal was skin followed by intestinal intubation and finally by gill infection.

Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent.

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.