Yolanda J. Brady

Catfish CC chemokines: genomic clustering, duplications, and expression after bacterial infection with Edwardsiella ictaluri

Chemokines are a family of structurally related chemotactic cytokines that regulate the migration of leukocytes, under both physiological and inflammatory conditions. CC chemokines represent the largest subfamily of chemokines with 28 genes in mammals. Sequence conservation of chemokines between teleost fish and higher vertebrates is low and duplication and divergence may have occurred at a significantly faster rate than in other genes. One feature of CC chemokine genes known to be conserved is genomic clustering. CC chemokines are highly clustered within the genomes of human, mouse, and chicken. To exploit knowledge from comparative genome analysis between catfish and higher vertebrates, here we mapped to bacterial artificial chromosome (BAC) clones 26 previously identified catfish (Ictalurus sp.) chemokine cDNAs. Through a combination of hybridization and fluorescent fingerprinting, 18 fingerprinted contigs were assembled from BACs containing catfish CC chemokine genes. The catfish CC chemokine genes were found to be not only highly clustered in the catfish genome, but also extensively duplicated at various levels. Comparisons of the syntenic relationships of CC chemokines may help to explain the modes of duplication and divergence that resulted in the present repertoire of vertebrate CC chemokines. Here we have also analyzed the expression of the transcripts of the 26 catfish CC chemokines in head kidney and spleen in response to bacterial infection of Edwardsiella ictaluri, an economically devastating catfish pathogen. Such information should pinpoint research efforts on the CC chemokines most likely involved in inflammatory responses.

Towards the Ictalurid Catfish Transcriptome: Generation and Analysis of 31,215 Catfish ESTs.

BackgroundEST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline information about highly expressed genes in various catfish organs to provide a guide for the production of normalized and subtracted cDNA libraries for large-scale transcriptome analysis in catfish.ResultsA total of 17 cDNA libraries were constructed including 12 from channel catfish (Ictalurus punctatus) and 5 from blue catfish (I. furcatus). A total of 31,215 ESTs, with average length of 778 bp, were generated including 20,451 from the channel catfish and 10,764 from blue catfish. Cluster analysis indicated that 73% of channel catfish and 67% of blue catfish ESTs were unique within the project. Over 53% and 50% of the channel catfish and blue catfish ESTs, respectively, had significant similarities to known genes. All ESTs have been deposited in GenBank. Evaluation of the catfish EST resources demonstrated their potential for molecular marker development, comparative genome analysis, and evaluation of ancient and recent gene duplications. Subtraction of abundantly expressed genes in a variety of catfish tissues, identified here, will allow the production of low-redundancy libraries for in-depth sequencing.ConclusionThe sequencing of 31,215 ESTs from channel catfish and blue catfish has significantly increased the EST resources in catfish. The EST resources should provide the potential for microarray development, polymorphic marker identification, mapping, and comparative genome analysis.

Communications: Bacteria in the Hemolymph of the Freshwater Prawn Macrobrachium rosenbergii

Abstract Bacteria from the hemolymph of the freshwater prawn Macrobrachium rosenbergii were identified and quantified. Total bacterial counts ranged from 0.0 to 4.6 × 105 cells/1.0 mL of hemolymph. Predominant bacteria isolated included Aeromonas spp., Bacillus sp., and Pseudomonas spp. No bacteria were found in the hemolymph of prawns without lesions. The predominant species of bacteria isolated from water samples of prawn culture ponds was a chitinoclastic Bacillus sp.

Preliminary Studies of a Newly Developed Subunit Vaccine for Channel Catfish Virus Disease

Abstract The soluble channel catfish virus (CCV) envelope was harvested and used as a vaccine for channel catfish virus disease. Three- to four-day-old eggs of channel catfish Ictalurus punctatus and 1-week-old fry were vaccinated by immersion. A booster was given to subgroups of fry 2 weeks after vaccination. Vaccinated and nonvaccinated control groups were challenged with viable CCV 8 weeks after vaccination. All challenged nonvaccinated control fry died during the first experiment, and 56% died in the second experiment. Survival offish vaccinated as eggs or fry was 31 and 82%, respectively; survival of groups given a booster dose was 81 and 89%, respectively.

Molecular biology of channel catfish virus

Abstract Channel catfish virus (CCV) is an important fish virus causing economic losses in areas where channel catfish ( Ictalurus punctatus ) is cultured. Although it has been almost three decade since the virus was isolated, molecular studies on CCV lagged behind other herpesviruses. Earlier studies dealt mostly with the basic biologic properties of CCV, and most of these studies focused on the pathogenecity and the detection strategies of the disease. Availability of the DNA sequence of CCV prompted many laboratories to study unique molecular characteristics of CCV to find its place in herpesvirus classification. The genomic structure of CCV was different enough so that CCV was given its own place within the herpesvirus classification. Unfortunately, some of the most important common characteristics of herpesviruses, such as gene regulation, latency, and virus–host cell interactions in CCV infection are not fully understood. Although different approaches have been taken to develop a vaccine to control CCV infections in farm raised catfish, these strategies have failed to find a product that could be used by farmers. This was mainly because of both applicability and the cost of the vaccine. Trends in the development of biotechnology, on the other hand, opened new windows to look into both molecular structure and the molecular cell-virus interactions and appear to be promising in explaining some of the fundamental questions in channel catfish virus disease.

In vivo gene expression of cold shock and other stress-related genes in Vibrio vulnificus during shellstock temperature control conditions in oysters

Aims:  To determine Vibrio vulnificus response to shellstock refrigeration conditions while the bacterium was embedded in oysters (in vivo).

Use of Turkey Meal as a Replacement for Menhaden Fish Meal in Practical Diets for Sunshine Bass Grown in Cages

Abstract A 391-d feeding trial was conducted with caged juvenile (mean weight = 36.2 g, SD = 8.04 g) sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis) to evaluate growth, feed conversion, protein efficiency ratio, body composition, and fillet composition resulting from diets with decreasing levels of fish meal (FM; 30, 20, 10, and 0%) and increasing levels of turkey meal (TM; 0.0, 9.7, 17.5, and 26.4%). We stocked 100 randomly selected fish into each of 12 floating cages (3.5 m3). Twice daily, fish were fed all they could consume in 30 min. Four practical diets were formulated to contain 40% protein and to be isoenergetic. After 391 d, significant (P ≤ 0.05) differences were found in growth; fish fed the control diet (30% FM, 0% TM) had higher individual final weight, percent weight gain, and specific growth rate than fish fed 10.0% FM and 17.5% TM. However, differences for those variables were not apparent between the control group and fish fed the 20% FM and 9.7% TM diet...

Gene expression of cold shock and other stress-related genes in Vibrio vulnificus grown in pure culture under shellstock temperature control conditions.

Shellstock refrigeration after harvesting is recommended to prevent further increases in Vibrio vulnificus numbers in oysters, but it could potentially induce a cold shock response in this bacterium. V. vulnificus was incubated at 35, 25, 20, and 15 degrees C and then subjected to 7.2 and 4 degrees C for 1 week. A cold-adaptation response that enhanced cell culturability was observed when cells were incubated at 15 degrees C prior to cold shock at 7.2 degrees C. In vitro cold shock gene expression was analyzed by reverse transcriptase PCR (RT-PCR). The expression of cold shock genes csp1 and csp5 (homologous genes to cspA and cspV) remained constant, despite cold shock. However, the transcript of csp3 was constitutively expressed before and after cold shock, with a few exceptions. The synthesis of csp3 mRNA in V. vulnificus C7184Tr (an avirulent strain) was induced only after 15 degrees C incubation and cold shock at 4 degrees C. The expression of csp4 was repressed after cold shock. Our data showed that the csp(s) tested in this study are not cold inducible. The transcripts of two oxidative stress-related genes, oxyR and katG, showed different induction patterns among strains after cold shock, suggesting that V. vulnificus cells encountered oxidative stress during cold shock.

Exposure of silver carp, Hypophthalmichthys molitrix, to Salmonella typhimurium

Abstract An experiment was conducted to determine if silver carp infected with salmonella could be depurated following transfer to clean water. A total of 140 silver carp was acclimated in static water, plastic swimming pools (7.3 m2 surface area) for 29 days. Pools were inoculated daily with a streptomycin-resistant strain of Salmonella typhimurium at a rale of 10 000 cells per liter of water. Thirty fish in lots of five fish each were examined for the presence of S. typhimurium in the kidney, mucous and intestine. Of these lots five contained salmonella in the intestine and one in the mucous and kidney. Infected fish were than transferred to clean static water, plastic pools for depuration. Thirty fish in lots of five each were sampled for salmonella in the mucous, kidney and intestine after being held for 4 and 14 days in clean water. Salmonella was isolated from the intestines of four lots. Silver carp were thus shown to be potential carriers of salmonella.

Communications: Viability of Bacterial Pathogens in Frozen Fish

Abstract Channel catfish Ictalurus punctatus injected with Aeromonas hydrophila, Pseudomonas fluorescens, Edwardsiella tarda, or E. ictraluri were frozen at −20°C after death. Bacterial isolation at 2-d intervals after freezing indicated that A. hydrophila could be recovered for 20 d, P. fuorescens for 60 d, E. tarda for 50 d, and E. ictaluri for 30 d in frozen fish.