Supplementary Methods and Figure 1 from The Novel Polyamine Analogue CGC-11093 Enhances the Antimyeloma Activity of Bortezomib

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

The United States (U.S.) response to viral hemorrhagic septicemia virus (VHSV) IVb emergence in the Laurentian Great Lakes (GL) included risk-based surveillance for cost-effective decision support regarding the health of fish populations in open systems. All U.S. VHSV IVb isolations to date derive from free-ranging fish from GL States. Most originate in the region designated by US Geological Survey hydrologic unit code (HUC) 04, with the exception of two detections in neighboring Upper Mississippi (HUC 05) and Ohio (HUC 07) regions. For States outside the GL system, disease probability was assessed using multiple evidence sources. None substantiated VHSV IVb absence using surveillance alone, in part due to the limited temporal relevance of data in open systems. However, Bayesian odds risk-based analysis of surveillance and population context, coupled with exclusions where water temperatures likely preclude viral replication, achieved VHSV IVb freedom assurance for 14 non-GL States by the end of 2012, with partial evidence obtained for another 17 States. The non-GL region (defined as the aggregate of 4-digit HUCs located outside of GL States) met disease freedom targets for 2012 and is projected to maintain this status through 2016 without additional active surveillance. Projections hinge on continued basic biosecurity conditions such as movement restrictions and passive surveillance. Areas with navigable waterway connections to VHSV IVb-affected HUCs (and conducive water temperatures) should receive priority for resources in future surveillance or capacity building efforts. However, 6 years of absence of detections in non-GL States suggests that existing controls limit pathogen spread, and that even spread via natural pathways (e.g., water movement or migratory fish) appears contained to the Great Lakes system. This report exemplifies the cost-effective use of risk-based surveillance in decision support to assess and manage aquatic animal population health in open systems.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

Cultured largemouth bass (LMB) cannot be sold as food in some lucrative markets due to regulatory restrictions that protect wild LMB. Distinguishing between cultured and wild fish could open food markets for cultured fish. Wild LMB eat freshwater fish and cultured LMB eat diets high in marine fish meal and oil, which should produce differences in flesh composition. We analyzed the proximate and fatty acid composition of wild and cultured LMB muscle to determine the potential for distinguishing fish origin analytically. Protein and moisture were higher in wild fish, while lipid was higher in cultured fish. The n-3 and n-6 fatty acids, long-chain polyunsaturated fatty acids (LC-PUFA), and the ratio of n-3 to n-6 fatty acids all differed between cultured and wild fish. The n-3 to n-6 ratio and n-3 LC-PUFA were higher in cultured fish, while elevated arachidonic acid (20:4n-6) in wild fish was a key distinguishing feature.

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼ 30-32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

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.

A new strain of Aeromonas hydrophila has been implicated in significant losses in farm-raised catfish. Outbreaks attributable to this new strain began in Alabama in the summer of 2009 and have spread to Arkansas and Mississippi in subsequent years. These outbreaks mostly afflicted market-sized fish and resulted in considerable losses in short periods of time. The present research was designed to develop an expeditious diagnostic procedure to detect the new strains of A. hydrophila due to the rapid onset and biosecurity concerns associated with this new disease. A discriminatory quantitative polymerase chain reaction assay was developed using gene sequences unique to the virulent strains identified in a related comparative genomic study. Using this assay, suspect colonies on a culture plate can be positively identified as the new strain within 2 hr. The assay is repeatable and reproducible with a linear dynamic range covering 8 orders of magnitude and a sensitivity of approximately 7 copies of target DNA in a 15-µl reaction. In addition, the assay is able to detect and quantify the virulent strain from catfish tissues (0.025 g), pond water (40 ml), and sediments (0.25 g) with a sensitivity limit of approximately 100 bacteria in a sample. This assay provides rapid discrimination between the new virulent strain and more common A. hydrophila and is useful for epidemiological studies involving the detection and quantification of the virulent strain in environmental samples and fish tissues.

Current US state and federal fish health regulations target the spread of viral hemorrhagic septicemia virus-IVb (VHSV-IVb) through movement restrictions of live fish; however, they largely ignore the potential for the virus to be spread through commercial distribution and use of frozen baitfish from VHSV-IVb-positive regions. Some state laws do require treatment of frozen baitfish to inactivate VHSV, and additional methods have been proposed, but few scientific studies have examined the efficacy of these treatments. In this study, bluegills Lepomis macrochirus were challenged with VHSV-IVb and frozen to represent standard industry methods, disinfected by various treatments, and tested for infectious VHSV-IVb using virus isolation. The virus was isolated from 70% of fish subjected to 3 freeze/thaw cycles. All other treatment methods were effective in inactivating the virus, including treatment with isopropyl alcohol, mineral oil, salt and borax, and dehydration. Dehydration followed by rehydration is rapid and effective, and therefore, seems to be the best option for inactivating VHSV-IVb present in frozen baitfish while maintaining their usefulness as bait.

A bacilliform virus was isolated from diseased fathead minnows (Pimephales promelas). Analysis of the complete genome coding for the polyprotein (pp1ab), spike (S), membrane (M) and nucleocapsid (N) proteins revealed that the virus was most like white bream virus (WBV), another bacilliform virus isolated from white bream (Blicca bjoerkna L.) and the type species of the genus Bafinivirus within the order Nidovirales. In addition to similar gene order and size, alignment of deduced amino acid sequences of the pp1ab, M, N and S proteins of the fathead minnow nidovirus (FHMNV) with those of WBV showed 46, 44, 39 and 15% identities, respectively. Phylogenetic analysis using the conserved helicase domain of the replicase showed FHMNV was distinct from WBV, yet the closest relative identified to date. Thus, FHMNV appears to represent a second species in the genus Bafinivirus. A PCR assay was developed for the identification of future FHMNV-like isolates.

Purpose: Enterotoxigenic Bacteroides fragilis (ETBF) are anaerobic enteric bacteria known to cause an acute, inflammatory diarrhea in both children and adults via its primary virulence factor the B. fragilis toxin (BFT). In murine models, ETBF causes asymptomatic, chronic colitis in C57BL/6 mice and increases colon tumorigenesis in multiple intestinal neoplasia (Min Apc+/-) mice. Prior human studies suggest an association between ETBF infection and active inflammatory bowel disease (IBD), though these studies are small in sample size. Our study aims to characterize the prevalence of ETBF colonization among pediatric control and IBD patients using combined anaerobic culture and fecal PCR diagnostic approaches. Methods: We obtained stool samples (n=259) and correlating clinical data from 105 prospectively enrolled pediatric patients with ulcerative colitis or Crohn's disease from two academic IBD centers. A single fecal sample was similarly collected from each of 105 confirmed non-IBD patients presenting to the same pediatric GI clinics. Frozen fecal samples were anaerobically cultured on Bacteroides Bile Esculin (BBE) agar to help promote Bacteroides spp. growth. Samples that did not grow on BBE or that grew poorly were plated on less selective Brucella anaerobic blood plates and/or LKV (Brucella Laked Blood Agar with Kanamycin and Vancomycin) plates in attempt to coax growth of Bacteroides spp. that may have been “stunned” by storage conditions. Any growth on Brucella blood agar or LKV plates was then transferred onto BBE. For all samples that grew on BBE, 16 single colonies were isolated and tested by touchdown PCR for the bft gene. Results: To date, 22 IBD samples have been tested. 17 grew on BBE, of which 10 samples contained isolates positive for bft by touchdown PCR (range of positive isolates per sample 1-8, average number of positive isolates per sample 3.6). In comparison, of the 14 control samples that were tested, 12 grew on BBE and 3 of these contained bft+ isolates (range 1-6, average 2.67). Conclusion: Our current data suggests that fecal carriage of ETBF is approximately twice as common in IBD patients compared to controls (45% vs. 21%, p=0.175). Analysis of our fecal bank is ongoing; updated microbiology results will be presented. We further plan to analyze the associated clinical data to determine if ETBF carriage correlates with clinical manifestations of IBD, such as disease location, severity, or timing of flares.

Members of the genus Francisella (viz., F. noatunensis subsp. orientalis [ Fno] and F. noatunensis subsp. noatunensis) have been described as causative agents of chronic granulomatous and pyogranulomatous lesions in wild and cultured fish species. In the present study, 68 archived formalin-fixed, paraffin-embedded (FFPE) tissues from several fish species, collected at different geographical locations from 2000 to 2011, were analyzed using a real-time polymerase chain reaction assay for the detection of the Fno intracellular growth loci C ( iglC) gene and by immunohistochemistry for the demonstration of Fno antigens. The results revealed a high correlation between these 2 diagnostic techniques validating their use for the diagnosis of Fno infection in archived FFPE tissues and confirming the presence of Fno in fish species from the Cari y years of the present century.

There has been a tremendous increase in global demand for marine and freshwater fish to meet the protein needs of our expanding human population. However, due to the limited capacity of the wild-capture sector and a levelling of production from capture fisheries, the practice of farming aquatic animals has expanded rapidly to become a major global industry. Aquaculture, particularly freshwater aquaculture is now integral to the economies of many countries. A large number of aquatic animal species are farmed in high density in freshwater, brackish and marine systems, where they are exposed to new environments and potentially new diseases. Further, environmental stress factors, the use of manufactured feeds, and prolific global trade has led to the emergence and spread of new diseases. Viral pathogens, established for decades or newly emerging as disease threats, are particularly challenging since there are few efficacious treatments. Vaccines have been developed for some viral fish pathogens in salmonids, but vaccines are not available for many of the viral pathogens important in Asia. Control and eradication programs are difficult because many viral infections remain latent until adverse environmental conditions, such as overcrowding or poor water quality, trigger the onset of disease. Here, we review the more significant viral pathogens of finfish in the Asia–Pacific including both those with a long history in Asian aquaculture and emerging pathogens including betanodaviruses and koi herpes virus that have caused massive losses in the freshwater aquaculture and ornamental fish industries.

There has been a tremendous increase in global demand for marine and freshwater fish to meet the protein needs of our expanding human population. However, due to the limited capacity of the wild-capture sector and a levelling of production from capture fisheries, the practice of farming aquatic animals has expanded rapidly to become a major global industry. Aquaculture, particularly freshwater aquaculture is now integral to the economies of many countries. A large number of aquatic animal species are farmed in high density in freshwater, brackish and marine systems, where they are exposed to new environments and potentially new diseases. Further, environmental stress factors, the use of manufactured feeds, and prolific global trade has led to the emergence and spread of new diseases. Viral pathogens, established for decades or newly emerging as disease threats, are particularly challenging since there are few efficacious treatments. Vaccines have been developed for some viral fish pathogens in salmonids, but vaccines are not available for many of the viral pathogens important in Asia. Control and eradication programs are difficult because many viral infections remain latent until adverse environmental conditions, such as overcrowding or poor water quality, trigger the onset of disease. Here, we review the more significant viral pathogens of finfish in the Asia–Pacific including both those with a long history in Asian aquaculture and emerging pathogens including betanodaviruses and koi herpes virus that have caused massive losses in the freshwater aquaculture and ornamental fish industries.

Smallmouth bass, Micropterus dolomieu Lacepède, bluegill, Lepomis macrochirus Rafinesque (coppernose strain), koi carp, Cyprinus carpio L., and channel catfish Ictalurus punctatus (Rafinesque), were infected by intraperitoneal injection with viral haemorrhagic septicaemia virus genotype IVb (VHSV-IVb) at 15 °C. When clinical signs of disease developed, one-third of the fish was moved to 20°C and one-third to 25°C. Mortality in challenged fish at all three temperatures ranged from 25 to 45% in smallmouth bass and from 70 to 90% in bluegill. No koi carp or channel catfish died during the study. Viral copy numbers detected by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in fish dying at 20 and 25°C decreased over time. In survivors of the challenge, viral copy numbers were higher in the more susceptible species (smallmouth bass and bluegill) than in the more VHSV-IVb disease-resistant species (koi carp and channel catfish). In fish surviving 28days post-infection, prevalence of infection was 66-100% depending on species and temperature, and VHSV-IVb was detected at 10(3) -10(5) copies μg(-1) host RNA. Our results show that qrt-RTPCR is a useful tool to investigate fish kills even 28days after temperatures are elevated above those known to be permissive for VHSV replication.

Pond-reared channel catfish Ictalurus punctatus with proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya spp., were examined with light and transmission electron microscopy to better characterize the inflammatory response during infection. The early stages of disease are characterized by the destruction of collagen in the matrix of the gill filament cartilage causing weakness and breaks within the gill filaments. These early lesions lacked a notable inflammatory response around the disrupted cartilage, a chondrocyte response was not apparent, and the parasite was not present, suggesting that the cartilage breaks occur prior to inflammation and arrival of the parasite in the gill. In later lesions, a significant inflammatory response was generated in areas of disrupted cartilage, and the inflammatory infiltrate was composed of a mixed population of granulocytes including neutrophils and cells that resembled eosinophils. The majority of eosinophil-like cells demonstrated evidence of degranulation. Trophozoites of Henneguya spp. were surrounded by a uniform population of cells believed to be neutrophils. The granulocytes were infiltrated within the dense collagen layer of the gill filament cartilage and often appeared within chondrocyte lacunae in place of the chondrocyte. The gill lamellae adjacent to the lesions were fused and contained an inflammatory infiltrate containing granulocytes and cells with pericentriolar granules that resembled previous descriptions of Langerhans-like cells. These cells were abundant within damaged lamellar epithelium, but were only rarely found within the gill filament. Lesions that appeared to be recovering lacked the dense collagenous layer around the cartilage and contained hyperplastic and hypertrophic chondrocytes that formed a callus. Other chondrocytes in the lesions had ultrastructural features indicative of cell death.