Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout. KEY WORDS: Rainbow trout · Streptococcus iniae · Serotype · Pathology Resale or republication not permitted without written consent of the publisherDis Aquat Org 62: [177][178][179][180] 2004 2003). In the present work we assessed the pathological outcomes of infections of rainbow trout by S. iniae serotype II. Our results indicate that Serotype II strains produce an acute septic disease accompanied by multisystem organ involvement. MATERIALS AND METHODSSampling procedure for pathology and bacteriological analysis. Specimens were collected from 2 trout farms located in northern Israel (Upper Galilee) which are supplied with water at a constant temperature of 16°C. At the time of collection both farms were experiencing heavy mortalities, and bacteriological examination (30 fish from each farm, 50 to 350 g each) revealed pure colonies of β-hemolytic Gram-positive cocci. Bacteriological identification (Eldar et al. 1995), authenticated by PCR analysis (Zlotkin et al. 1998), confirmed the presence of arginine dehydrolase-negative isolates of Streptococcus iniae. PCR analysis with the p14 primer did not produce the 750 bp band, indicating that all current isolates were of type II (Bachrach et al. 2001). Tissues (brain, heart, spleen, kidney, liver, intestine, gills and muscle) from 10 diseased rainbow trout were fixed in 10% neutral buffered formalin and stained with hematoxylin and eosin (HE). Gross lesions were recorded.Distribution of bacteria in tissues. Bacterial colonyforming units (CFUs) were counted in the tissues of 20 rainbow trout (100 to 200 g each), infected with Streptococcus iniae type II and showing clinical signs of streptococcosis (darkening of the skin combined with multifocal ecchymoses, lethargy, loss of orientation and ocular pathologies). Organs (brain, heart, spleen, kidney, liver, intestine, gills and muscle) were weighed and blended over a 60-mesh grid. Tissue homogenate samples (1 g) were suspended in 10 ml phosphatebuffered saline (PBS) (15 mM Na 2 HPO 4 , 145 mM NaCl, pH 7.20) supplemented with 0.2% Triton X-100 (Sigma) and serially diluted. The bacterial CFU population was determined by plate counting.The bacterial loads in tissues of rainbow trout infected by Streptococcus iniae type I were assessed with a 2-step cohabitation method. First, 10 naive rainbow trout (50 to 80 g each) were infected by intraperitoneal inoculation of (7.5 × 10 5 CFU fish -1 ) virulent S. iniae Dan-15 (type I strain), as described previously (Eldar et al. 1997a). Then, on Day 10 ...
By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.
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