A newly recognized herpesvirus, koi herpesvirus or KHV, causes a lethal disease in common carp, Cyprinus carpio, and its colourful strain known as koi or fancy carp. In this study, we report new outbreaks of the disease, present initial characterization of the KHV genome, and describe assays for detection of KHV DNA in infected cells and tissues of infected fish. Restriction endonuclease (RE) profiles of viral DNA derived from two epidemiologically distinct KHV isolates were identical to each other. Cloned KHV BamHI and SphI DNA probes specifically hybridized to KHV DNA, but not to DNAs derived from a variety of other fish herpesviruses. The KHV DNA probes detected KHV DNA in tissues of experimentally infected koi fish by DNA hybridization. The KHV specific polymerase chain assays (PCR) were developed for rapid detection and confirmation of KHV DNA in tissues of infected fish.

Lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1) is an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium. Here we analyze the organization of the Lu-ECAM-1 protein complex, report the sequence of Lu-ECAM-1 cDNAs, and reveal a novel function of the protein. Lu-ECAM-1 immunopurified from bovine aortic endothelial cells (BAEC) consists of tightly associated glycoproteins of 90, 38, and 32 kDa, with minor components of 130 and 120 kDa. We present evidence that all of these protein species are encoded by a single open reading frame whose initial translation product is proteolytically processed to yield the other products. Correct processing in vitro was demonstrated by transfection of the longest cDNA into human embryonic kidney 293 cells; immunoblot analysis showed that the ϳ120-kDa precursor gave rise to 90-and 38-kDa products. RNA blots of BAEC mRNA detected messages in agreement with the sizes of the cDNA clones in addition to several of high molecular weight. DNA blot analysis showed that Lu-ECAM-1 is conserved throughout its length in all mammals tested, usually as a single or low copy gene. In the bovine, Lu-ECAM-1 protein is 88% identical to a calcium-dependent chloride channel described recently in tracheal epithelium, Ca-CC. Probes for Lu-ECAM-1 mRNA and protein confirmed the presence of a homolog in this tissue. We show that messages for both proteins are present in lung while only Ca-CC is present in trachea and only Lu-ECAM-1 is present in BAEC. These results suggest that endothelial cells express a chloride channel that is related to, but distinct from, that expressed in tracheal epithelium. They further suggest that an adhesion molecule can also be a chloride channel.The preference of metastasizing tumor cells for certain organs may be explained if such cells fortuitously recognize and adhere to organ-specific, endothelial cell-surface molecules. In studying this hypothesis, much emphasis has been placed on the role of members of the classic families of cell-cell adhesion molecules including selectins, the immunoglobulin superfamily, and integrins (1-3). The contribution of these adhesion molecules to organ preference of metastasis was suggested by a number of reports describing the presence of such molecules on endothelia of various tissues and vessel calibers and denoting corresponding ligands on malignant cells of tumors of various tissue origins (1-7). In our laboratory, a different approach was chosen to testing the contribution of specific endothelial cell adhesion molecules to organ preference of metastasis. It relied on an endothelial cell culture system that could be modulated by growing unspecific, large vessel-derived endothelial cells (e.g. bovine aortic endothelial cells (BAEC) 1 ) on organ-specific matrix to express phenotypic traits of the microvasculature of that organ (8, 9). Tumor cells with distinct metastatic dissemination patterns were then evaluated for adhesion to these endothelial cells, observing that tumor cells only...

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To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Gills of 194 fountain darters Etheostoma fonticola collected from the Comal River in Texas from May 1997 through May 1998 were found to be parasitized with 8-1,524 metacercarial cysts of a heterophyid trematode tentatively identified as Centrocestus formosanus. The intensity of infection varied among three sites on the Comal River. In contrast, of 130 darters from the nearby San Marcos River that were examined, only 4 (3%) were infected, and these had 1-2 cysts per fish. Of 2,279 Melanoides tuberculata snails from the Comal River that were examined, 139 (6.1%) were infected with the trematode. Only 1 snail in 2,241 from the San Marcos River that were examined was infected. The presence of metacercariae in darters was associated with flared opercula, shortened or thickened gill filaments, epithelial hyperplasia, and engorged lamellae. The normal cartilage support of the filaments was distorted and displaced, leading to severe deformities of filament structure. Gill damage was severe and possibly life threatening for the darters with more than 800 cysts per fish (9% of examined fish). We suspect that fountain darter deaths were caused by the parasite in the Comal River during this study.

Moribund goldfish Carassius auratus from private collections and breeding facilities were tested for hematopoietic necrosis herpesvirus 2 (also known as cyprinid herpesvirus 2 [CyHV-2]) by degenerate polymerase chain reaction (PCR) of the viral polymerase gene followed by sequencing. The degenerate PCR method produced a fragment with a DNA sequence that was more than 99% identical to the sequence of CyHV-2. Histology and electron microscopy showed that the moribund fish had severe necrosis in the hematopoietic tissues of the kidney and spleen and that herpesvirus particles were present. In the three cases studied, the presence of the virus was associated with high mortality (up to 80%) that could not be attributed to any other cause. The CyHV-2 is very difficult to isolate in cell culture, but our PCR and case studies demonstrate that this virus has a wide geographic distribution in the United States and that goldfish hematopoietic necrosis herpesvirus disease is likely to be an important, but rarely detected, disease of goldfish.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen of goldfish Carassius auratus auratus L. that causes herpesviral hematopoietic necrosis (HVHN) disease. The disease is associated with necrosis of hematopoietic tissues and anemia with high mortality. We have developed a real time 5'-nuclease PCR method (Taqman) that quantitatively detects CyHV-2 with a linear response over 8 logs of target concentration. The coefficient of variability on replicate samples tested on different days was 13% and the calculated sensitivity approached 1 target molecule per reaction. The assay does not cross-react with other similar fish herpesviruses, including CyHV-1 (carp pox) and CyHV-3 (koi herpesvirus), but reliably detects known CyHV-2 positive fish. The assay detects CyHV-2 not just in clinical cases of HVHN but also in apparently healthy 1 yr old goldfish fingerlings and even in 3 to 5 yr old broodfish.

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.