Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders.
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We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91 %) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions. (+info)
Results from black tiger shrimp penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV).
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Commercial, intensive, earthen shrimp ponds (188) in southern Thailand were stocked with postlarvae (PL) of Penaeus monodon that had tested positive or negative for white-spot syndrome virus (WSSV) infection by polymerase chain reaction (PCR) assay. All the PL were grossly healthy. At 2 wk intervals after stocking, shrimp from each pond were examined for gross WSSV lesions and tested for WSSV by PCR. Shrimp from all the ponds stocked with WSSV-PCR-positive PL (Group 0, n = 43) eventually showed gross signs of white-spot disease (WSD) at an average of 40 d after stocking. Of the remaining ponds stocked with WSSV-PCR-negative PL (n = 145), some remained WSSV-PCR-negative throughout the study (Group 5, n = 52), while others (93) became WSSV-PCR-positive after stocking, during the first month (Group 1, n = 23), second month (Group 2, n = 40), third month (Group 3, n = 24), or fourth month (Group 4, n = 6). Crop failure was defined as a pond drain or forced harvest before 14 wk or 98 d of cultivation. For Group 0 the proportion of ponds failing was 0.953, while it was only 0.019 for Group 5. Thus, the relative risk of failure for Group 0 was approximately 50 times that of Group 5. The relative risk of failure for Group 0 was also 3 times that for ponds stocked with WSSV-PCR-negative PL. Obviously, not all WSSV outbreaks resulted in crop failure. Of the 93 ponds stocked with PCR-negative PL that later yielded WSSV-PCR-positive shrimp, 53% reached successful harvest. The study showed that PCR screening of PL and rejection of WSSV-positive batches before stocking could greatly improve the chances of a successful harvest. (+info)
A herpes-like virus infects a non-ostreid bivalve species: virus replication in Ruditapes philippinarum larvae.
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Sporadic high mortalities were reported in June 1997 among hatchery-reared larval Manila clam Ruditapes philippinarum in a French commercial hatchery. Cellular abnormalities were observed in semi-thin sections in affected animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in larvae. This is the first description of a herpes-like virus infection in larval R. philippinarum, a non-ostreid bivalve species. Virus particles were similar to other herpes-like viruses described from different oyster species with respect to ultrastructure and morphogenesis. Electron microscopic examination also demonstrated cells with condensed chromatin and extensive perinuclear fragmentation of chromatin. Like viruses infecting oysters, the herpes-like virus detected in clams may induce apoptosis in infected animals. (+info)
Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.
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Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE). (+info)
Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 16S rDNA sequencing.
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An epizootic bacterial infection in the giant freshwater prawn Macrobranchium rosenbergii occurred in Taiwan from May to June 1999. The cumulative mortality was approximately 30 to 75%. The diseased prawns showed opaque and whitish muscles and were approximately 2 mo old with total lengths from 5 to 6 cm. Histopathologically, they showed marked edema and necrotic lesions with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Three isolates from diseased prawns at different farms were tested using the API 20 Strepsystem and conventional tests and identified as Lactococcus garvieae. Experimental infections with these isolates gave gross signs and histopathological changes similar to those seen in the naturally infected prawns. The LD50 value of isolate MR1 was 6.6 x 10(5) colony forming units/prawn. Identification of MR1 was confirmed by a PCR assay for L. garvieae that gave the expected amplicon of 1100 bp. In addition, its 16S rDNA sequence (GenBank accession number AF283499) gave 99% sequence identity to Enterococcus seriolicida (synonym L. garvieae; GenBank accession number AF061005). This is the first report of confirmed L. garvieae infection in prawn aquaculture. (+info)
Characterisation of Saprolegnia sp. isolates from channel catfish.
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Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups. (+info)
Effects of bacteria on the growth of an amoeba infecting the gills of turbot.
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We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination. (+info)
Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile.
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The isolation of infectious salmon anaemia virus (ISAV) from asymptomatic wild fish species including wild salmon, sea trout and eel established that wild fish can be a reservoir of ISAV for farmed Atlantic salmon. This report characterizes the biological properties of ISAV isolated from a disease outbreak in farmed Coho salmon in Chile and compares it with ISAV isolated from farmed Atlantic salmon in Canada and Europe. The virus that was isolated from Coho salmon tissues was initially detected with ISAV-specific RT-PCR (reverse transcription-polymerase chain reaction). The ability of the virus to grow in cell culture was poor, as cytopathology was not always conspicuous and isolation required passage in the presence of trypsin. Virus replication in cell culture was detected by RT-PCR and IFAT (indirect fluorescent antibody test), and the virus morphology was confirmed by positive staining electron microscopy. Further analysis of the Chilean virus revealed similarities to Canadian ISAV isolates in their ability to grow in the CHSE-214 cell line and in viral protein profile. Sequence analysis of genome segment 2, which encodes the viral RNA polymerase PB1, and segment 8, which encodes the nonstructural proteins NS1 and NS2, showed the Chilean virus to be very similar to Canadian strains of ISAV. This high sequence similarity of ISAV strains of geographically distinct origins illustrates the highly conserved nature of ISAV proteins PB1, NS1 and NS2 of ISAV. It is noteworthy that ISAV was associated with disease outbreaks in farmed Coho salmon in Chile without corresponding clinical disease in farmed Atlantic salmon. This outbreak, which produced high mortality in Coho salmon due to ISAV, is unique and may represent the introduction of the virus to a native wild fish population or a new strain of ISAV. (+info)