gTilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide.

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The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that ϳ70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.The fish pathogen Streptococcus iniae (11) is endemic in various parts of the world, including Israel (6) and North America (10). S. iniae infection of rainbow trout (Oncorhynchus mykiss) produces a disease which substantially affects the brain, with only minor pathological changes in other organs (4). Recently, S. iniae has been isolated from diseased humans suffering from cellulitis, meningitis, and bacteremias, indicating a threat to public health (16).A specific S. iniae vaccine became available in 1995. From 1995 to 1997, all Israeli trout farms in the Upper Galilee, (which share water reservoirs) routinely vaccinated their entire stocks (roughly 3 million fish/year), reducing S. iniae-related mortalities from 50% annually to less than 5% (5). However, massive new outbreaks of the disease were recorded in 1997. Unlike the previous pathological manifestations, diseased fish exhibited multisystem organ involvement and diffuse internal hemorrhages. Brains samples were collected from diseased fish and streaked on Columbia agar base (Difco) supplemented with 5% (vol/vol) defibrinated sheep blood. Beta-hemolytic gram-positive cocci were detected following an incubation of 24 to 48 h at 24°C. Conventional identification schemes (API 20 STREP; BioMerieux SA, Marcy l'Etoie, France) suggested that all isolates (of 100 collected over 24 months) were S. iniae. The new isolates, unlike the previous isolates, were shown to be arginine dehydrolase (ADH) negative. Definitive identification was accomplished by PCR, using S. iniae 16S rDNAspecific primers (Zlotkin et al. [17]), which revealed the 300-bp S. iniae-specific PCR product in all isolates.Six (11)). The rDNA sequence analyses confirmed that the six isolates were S. iniae. All had an identical sequence (GenBank accession no. AF335573) and differed from that of S. iniae ATCC 29178 (GenBank accession no. AF335572) in six bases (99.6% homology). EcoRI and HindIII digests of DNAs extracted from early and recent isolates resulted in identical restriction fragment length polymorphism ribotype patterns (data not shown), indicating that all isolates cluster in the Israeli S. iniae rank (6). The use of additional endonucleases (PvuII or KpnI) did not provide strain-to-strain differentiation (data not shown).The random amplified polymorphic DNA (RAPD) technique ( Fig. 1) was used to distinguish early from recent isolates. Primer p14 (5ЈGATCAAGTCC), previously proven useful for discrimination among group A streptococcol strains, was used (Neeman et al. [8]). All early (1991 to 1995) isolates produced a band with an estimated length of 750 bp. No such band was found in the PCR product of the recent isolates (Fig. 1).Hyperimmune sera for serological differentiation were obtained by three monthly immunizations of rainbow trout with formalin-fixed S. inia...

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Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.

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 ...