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 TCID 50 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. KEY WORDS: Piscirickettsia salmonis · Pathogenesis · Fish disease · Coho salmonResale or republication not permitted without written consent of the publisher

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial-free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE-214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis.

In the last 9 years, epizootics of an icterus condition has affected coho salmon, Oncorhynchus kisutch (Walbaum), reared in seawater cages in southern regions of Chile. At necropsy, fish from field cases exhibited signs of jaundice accompanied by pale light-brown livers and dark spleens. Histopathological and haematological results indicated that these fish presented haemolytic anaemia. After microbiological examination no bacterial or viral agents could be identified as aetiological agents of this disease. In an infectivity trial, coho salmon, Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), were inoculated intraperitoneally with a filtrate of an organ homogenate (0.45 microm) from a diseased coho salmon and held for 60 days in tanks supplied with fresh water. The disease was only reproduced in coho salmon in which mortalities, beginning at day 23 post-inoculation (p.i.), reached a cumulative value of 24% at day 27 p.i. This condition was transmitted to non-inoculated cohabiting coho salmon suggesting that it is a waterborne disease. Thus, this icteric condition is caused by an infectious form of haemolytic anaemia, probably of viral aetiology, and coho salmon are more susceptible than either Atlantic salmon or rainbow trout.

Piscirickettsia salmonis is an obligate bacterial pathogen which causes piscirickettsiosis, a systemic disease affecting some anadromous and marine teleost fish species. To investigate the pathogenesis of this disease, a time-course study was conducted, using immunohistochemistry, after challenging rainbow trout Oncorhynchus mykiss (Walbaum) fry by an immersion bath with P. salmonis.To carry out this assay, fish (total n = 72; weight % 2.5 g) were allotted to six subgroups (12 fish each) held in individual 50-L tanks supplied with a flow-through freshwater system (25 L h À1 ) at 15.4°C (SD 0.8).The SLGO-95 strain of P. salmonis (Smith et al. 1996b) was used. Bacteria, after thawing, were cultured and titered, by end-point dilution assay, in the CHSE-214 cell line. Cells were cultured at 18°C in Eagle's minimal essential medium with Earle's salts (MEM), which is autoclavable (MEM Auto-Mod Sigma-Aldrich), supplemented with 2 mM L-glutamine and 10% of foetal calf serum (both from Gibco, Life Technologies).For the experimental challenge, four fish subgroups (A 1 , A 2 , B 1 and B 2 ) were exposed to P. salmonis, each one as an independent batch, by immersion for 15 min in a 50-mL bacterial suspension (in MEM) containing %10 5 tissue culture infectious dose 50% per mL. After exposure, each subgroup was kept for 30 min in 1-L sterile MEM, although some fish were sampled within this period, and was then replaced in the 50-L tanks. Fish from subgroups A 1 and A 2 were used to record clinical signs, mortalities and gross and histological lesions (standard haematoxylin and eosin) for 30 days after the immersion challenge, as a bacterial virulence control. Methanol-fixed smears from the kidneys of dead fish were obtained for Gram staining and also for labelling to detect P. salmonis by the indirect immunofluorescence test (IFAT) according to Lannan, Ewing & Fryer (1991). Two additional fish subgroups (C 1 and C 2 ) were included as non-infected controls. Excepting that these fish were sham-exposed, they were treated as subgroups A 1 and A 2 . All these immersion procedures were carried out under permanent aeration at 16.5 (AE0.5)°C.Fish of subgroups B 1 and B 2 were sequentially sampled. Three fish were killed by anaesthetic overdose (Tricaine Sigma-Aldrich) at each of the following post-exposure (post-exp) times counted from the start of the immersion with P. salmonis: 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 18 h and 3 days. Fish were formalin-fixed and processed by standard histological methods. Five-lm sagittal sections of the whole body, excepting the tail, were examined, using an immunoperoxidase test to detect P. salmonis in the tissues.

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To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post-exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post-clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC-1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.