Streptococcus shiloi strains, including the type strain, which were isolated in Israel and the United States, and Streptococcus iniae ATCC 29178T (T = type strain) are phenotypically identical (as determined with API 20 STREP and API 50CH kits; beta-hemolytic on sheep blood agar). DNA-DNA hybridization experiments revealed levels of homology of 77 to 100%. Thus, S. shiloi should be considered a junior synonym of S. iniae. This bacterium is a major fish pathogen that is distributed worldwide.Streptococcus iniae was described in 1976 by Pier and Madin (7), who isolated this bacterium from skin lesions of a captive Amazon freshwater dolphin (Inia geofiensis). In 1994, we described a new streptococcal species that was isolated from diseased rainbow trout (Onchorynchus mykiss) in Israel and was named Streptococcus shiloi (2). This name was validated in 1995 (4). S. shiloi differed from S. iniae in G + C content (37 mol% [2]; the G + C content of S. iniae is 32.9 mol% [7]), pathogenicity and host range. S. shiloi was shown to be the etiologic agent of an acute meningoencephalitis that affects trout (3), and it was also shown to be the causative agent of a similar disease in tilapia (Oreochromis aurea X Oreochromis nilotica hybrids) along with Streptococcus dificile (2). S. shiloi and S. dificile were shown to have broad geographical distributions and broad host ranges (2). S. shiloi was also isolated from diseased tilapia in Taiwan and the United States, and S. dificile was isolated in Japan from sea-cultured yellowtails (Seriola quinqueradiata) (2).The purposes of this work were to elucidate the taxonomic position of S. shiloi and to compare S. shiloi Israeli field isolates collected over the last 5 years with isolates collected in the United States during the last 2 years.A total of 15 American isolates were compared with 19 Israeli isolates, including the type strain of S. shiloi (strain ND 2-16 [= CIP 103769]), 1 strain isolated in Taiwan, and S. iniae ATCC 29178T (T = type strain) ( Table 1). All of the strains were isolated from diseased fish. The type of hemolysis was determined on Columbia agar base (Difco) supplemented with 5% (vol/vol) defibrinated sheep blood; the type of hemolysis produced on this medium was then compared with the type of hemolysis produced on the same medium supplemented with human or bovine blood. Biochemical reactions were determined with API 20 STREP and API 50CH systems (API, La Balmes Les Grottes, France). Most of the instructions of the manufacturer were followed; the only exception was the temperature of incubation, which was adjusted to 30°C. Results were read after 24 h of incubation. DNA was extracted by a previously described method (2). DNA-DNA hybridization experiments were performed by using the hy- l), except that the volumes used were modified (2). After denaturation by boiling, reassociation was allowed to proceed for 18 h at 60°C. Duplicate reactions were performed, and each run was performed twice. The levels of DNA relatedness (relative binding ratios) and the differe...
We analysed 20 boxes of, frozen imported bait-shrimp (China: Parapenaeopsis sp. and Metapenaeopsis sp.) and 8 boxes of native, frozen bait-shrimp (Gulf of Mexico: Litopenaeus setiferus and Farfantepenaeus duorarum) by RT-PCR or PCR for Taura syndrome virus (TSV), yellowhead virus/gill-associated virus (YHV/GAV), white-spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV). All 28 boxes of shrimp were negative for TSV, YHV/GAV and IHHNV; 2 boxes of imported bait-shrimp were WSSV-positive by 3 different PCR assays. Intramuscular injection of replicate groups of SPF (specific pathogen-free) L. vannamei juveniles with 2 different tissue homogenates prepared from the 2 WSSV-positive bait boxes resulted in 100% mortality of the test shrimp within 48 to 72 h post-injection. No mortality occurred among injected negative control groups. Histological and in situ hybridization analyses of 20 moribund treatment-shrimp demonstrated severe WSSV infections in each sample. Oral exposure of SPF L. vannamei postlarvae, PL (PL 25 to 30 stage; ~0.02 g) to minced tissue prepared from the 2 WSSVpositive bait-lots did not induce infection, possibly because of an insufficient infectious dose and/or viral inactivation resulting from multiple freeze-thaw cycles of the bait-shrimp during PCR testing. Use of an electric drill and collection of drill-tailings (tissue from ~20 to 30 shrimp) from frozen blocks of shrimp was successfully employed as an alternate tissue-sampling method without thawing. Our findings indicate that imported WSSV-infected bait shrimp, originating from China, are being sold in Texas for the purpose of sport fishing and represent a potential threat to freshwater and marine crustacean fisheries, as well as to coastal US shrimp farms.KEY WORDS: White-spot syndrome virus · PCR · Histopathology · Bait-shrimp · Disease transmission · Biosecurity · Penaeid shrimp Resale or republication not permitted without written consent of the publisherDis Aquat Org 71: [91][92][93][94][95][96][97][98][99][100] 2006 variably staining intranuclear inclusion bodies within multiple target tissues that are readily identifiable by light microscopy and are considered pathognomonic for this disease (Lightner 1996).The first recorded outbreak of WSSV in the western hemisphere occurred among wild-caught Gulf of Mexico (GOM) Litopenaeus setiferus shortly after they were stocked in a South Texas shrimp farm in 1995 (Lightner 1996). Between late 1995 through early 1997, multiple WSSV epizootics were documented among feeder crayfish populations (Orconectes punctimanus and Procambarus sp.) at the National Zoo in Washington DC (Richman et al. 1997). Chang et al. (2001) demonstrated the presence of asymptomatic WSSV infections among wild Atlantic blue crabs Callinectes sapidus collected off the coasts of New York, New Jersey and Texas during 1997. Subsequent WSSV detection among indigenous GOM L. setiferus or Farafantepenaeus aztecus stocks occurred in , 2001 (pers. comm. P. Ostrowski, T. Varner, CE...
Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease that was previously identified solely in cultured Penaeus vannamei, the Pacific white shrimp, from Texas (USA). In January 1993, a disease with similar clinical and histopathologic features to NHP was diagnosed in P vannamei cultured in Peru. Oligonucleotide primers derived from variable regions V5, V8 and V9 of the 16s ribosomal RNA gene sequence enabled polymerase chain reaction (PCR) confirmation of the diagnosis of NHP in both Texan and Peruvian shrimp. The PCR amplification products from the clinical specimens were compared to the PCR amplification product obtained from a sucrose-gradient purified sample of bacteria that was previously used to reproduce NHP. In this study, fingerprinting by restriction fragment length polymorphism (RFLP) analysis of the PCR products was used to compare the isolates. Results indicate that hepatopancreatic lesions are caused by the same bacterium in both Texas and Peru. Negative results were obtained from uninfected shrimp and shrimp infected with Vibr~o spp., as well as DNA extracted from Brucella abortus, Ehrlichia risticji, SalmoneUa enteritidis, Agrobacterium tumefaciens and Streptococcus crlae.
This is the first report of natural white spot syndrome virus (WSSV) infection in wild and large-scale farmed crawfish. In the spring of 2007, 3 crawfish farms experienced heavy mortality in ponds populated by Procambarus clarkii and P. zonangulus. Histological examination revealed findings consistent with severe viral infection characterized by numerous intranuclear inclusions in ectodermal and mesodermal tissues. Samples tested by in situ hybridization, injection bioassay in Litopenaeus vannamei, and PCR (nested and real time) were all positive for WSSV. Samples were sent to the National Veterinary Services Laboratory in Ames, Iowa, USA, where WSSV was verified. Subsequently, a multi-parish survey of 184 sites in Louisiana (including farm and wild basin samples) using real-time PCR determined that > 60% of sites sampled were positive for WSSV, including wild basin samples.
Presumptive systemic streptococcal infections were detected histologically in farmed Litopenaeus vannamei juveniles submitted from a Latin American country and the bacteria isolated. Characterization work demonstrated that the Gram-positive cocci form chains, grow aerobically and anaerobically, are oxidase-and catalase-negative, non-hemolytic, non-motile, Lancefield Group B positive and PCR positive when amplified with a universal streptococcal primer set. Differing Streptococcus identifications were obtained using API 20 Strep and Biolog systems, the former identifying the isolate as S. uberis and the latter as S. parauberis. Injection of specific pathogen-free (SPF) L. vannamei with the bacteria resulted in 100% mortality by 3 d post-injection with successful recovery of the agent from moribund test shrimp hemolymph samples. The recovered isolate was used in per os and waterborne exposure studies of SPF L. vannamei with mortalities ranging from 40 to 100% and 80 to 100%, respectively. Histologic analysis of 5 to 8 moribund shrimp from each exposure method demonstrated that all contained a severe bacteremia characterized by numerous free cocci within the hemolymph and aggregates of vacuolated hemocytes with notable intravacuolar cocci. This unique lesion type was most pronounced within the lymphoid organ and considered pathodiagnostic for this disease. Experimentally induced lesions were identical to those in naturally infected farmed shrimp and the Streptococcus sp. responsible was re-isolated, fulfilling Koch's postulates. Five freeze/thaw cycles of 10 experimentally infected shrimp were performed over a 2 mo period and the bacteria successfully cultured from all shrimp at each interval. These collective findings describe the first reported case of streptococcosis in marine penaeid shrimp in the Western Hemisphere and indicate that the agent may be disseminated via live or frozen infected shrimp.
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