Phytogenic feed-additive effects on channel catfish rhamnose-binding lectin levels, and susceptibility to Edwardsiella ictaluri.

We investigated the effects of a phytogenic feed additive on disease susceptibility to Edwardsiella ictaluri in channel catfish Ictalurus punctatus and regulation of 6 rhamnose-binding lectin (RBL) genes. Juvenile catfish (n = 250, 13.4 ± 0.1 g) were allotted to the following treatments: control (floating diet) or EO (floating diet supplemented with essential oils; Digestarom® P.E.P. MGE). The fish were fed their respective diets for 6 wk. Following subjection to different feed treatments, all fish were exposed to pathogenic E. ictaluri by bath immersion. Another group of fish were not challenged (non-challenged controls, fed control feed). Mucosal tissue samples were taken to quantify gene expression levels of RBL on Days 1 and 2 post-challenge. After challenge, survival was higher (64.4 vs. 48.0%) in fish fed EO compared to controls (p < 0.05). Relative to non-challenged controls, gill RBL1a mRNA was higher in fish fed EO (p < 0.05) on Day 1 while gill RBL3b was higher in fish fed EO (p < 0.01) on Days 1 and 2, respectively. RBL5a in the skin and proximate small intestine did not change significantly relative to non-challenged fish on Days 1 and 2 of the disease challenge. Results demonstrate that Digestarom® P.E.P. MGE improved survival of channel catfish challenged with E. ictaluri. One of the mechanisms through which essential oils may improve survival is through upregulation of RBL1a and RBL3b in the gill.

Effects of a phytogenic feed additive on growth performance, susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin.

A study was conducted to investigate the effect of a phytogenic feed additive (Digestarom® P.E.P. MGE; containing the essential oils carvacrol, thymol, anethol, and limonene) on growth performance and disease susceptibility to Edwardsiella ictaluri. Two hundred and fifty juvenile channel catfish, Ictalurus punctatus (7.2 ± 0.1 g) were allotted into the following treatments: Control (floating diet) and EO (floating diet supplemented with essential oils). The fish were fed their respective diets for 6 weeks. At the end of the study, all fish were exposed to virulent E. ictaluri by bath immersion (1.9 × 10(7) cfu/mL; final concentration). Plasma and tissue samples were taken to quantify protein and mRNA expression levels of mannose binding lectin (MBL). Weight gain and food conversion ratio were similar between treatments. After exposing fish to virulent E. ictaluri and monitoring mortality for 21 days, survival was 43% higher (69.5 vs 48.4%) in fish fed EO compared to fish not treated with EO (P < 0.05). One day after challenge, plasma MBL levels were down-regulated in the non-treated fish compared to non-challenged fish. In the EO fish, MBL levels were similar to non-challenged fish but significantly higher than non-treated fed fish (P < 0.001). By d 7, plasma MBL levels increased in non-treated fed fish to levels observed in the EO and non-challenged fish. On d 14, MBL mRNA levels were upregulated 15-fold in fish fed EO compared to non-treated fed fish and non-challenged fish (P < 0.001). The results demonstrate that essential oils improved survival of channel catfish challenged with E. ictaluri. Mechanisms through which essential oils improve survival may involve MBL.

Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P < 0.01). Expression of UCP2b mRNA tended to be lower (P < 0.10) in fast growing fish in the middle of the study although levels were similar at the beginning and the end of the study. In the fed vs fasted study, expression of UCP2b mRNA in muscle was increased (P < 0.05) in fish assigned to 30 d of fasting. Our results suggest that, based on the nucleotide and amino acid sequence similarities and tissue mRNA distribution, catfish UCP2b may be the analog to UCP3. Moreover, our results suggest selection toward growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms.

Histological and computed tomographic evaluation of a parasitic conjoined twin in hybrid catfish (Ictalurus punctatus [Rafinesque] X Ictalurus furcatus [Lesueur]).

There is growing use of hybrid catfish (Ictalurus punctatus ♀ X Ictalurus furcatus ♂) in commercial aquaculture to utilize hybrid vigour to improve production A conjoined twin specimen found during the course of production studies by the United States Department of Agriculture Catfish Genetic Research Unit (USDA-CGRU) was submitted to the Aquatic Research and Diagnostic Laboratory (ARDL). After preliminary inspection, it was transported to Mississippi State University, College of Veterinary Medicine for further evaluation. The specimen was examined using both computed radiography and computed tomography antemortem. Following humane euthanasia, the specimen was examined both grossly and histologically. Tissues from both fish were also submitted for genetic analysis to determine whether twins were derived from the same egg. This report records the presentation and examination of a pair of conjoined hybrid catfish (I. punctatus X Ictalurus furcatus).

Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

A standardized microsatellite marker panel for parentage and kinship analyses in channel catfish, Ictalurus punctatus.

This research was designed to produce a standardized set of microsatellite loci for parentage and kinship analyses in channel catfish, the leading species of US aquaculture. Three panels of five to six markers each were developed that contained a total of two dinucleotide-, eight trinucleotide- and seven tetranucleotide-microsatellite loci respectively. The loci had a range of nine to 31 alleles per locus in an outbred population. Based on the allele frequencies measured in commercial randomly bred broodstock, the combined probability of non-exclusion of an unrelated candidate parent pair was 5.36e-18. The combined probability of non-exclusion of unrelated identical genotypes was 2.58e-08. The microsatellite panels were validated by parentage and kinship evaluation in three populations. A total of 697 spawns were collected from matings of outbred broodstock over three spawning seasons, and parents were determined unambiguously for all but three spawns. Genotype analysis also enabled the identification of half-sibling and full-sibling families produced by pond spawning. In a second experiment, parentage was unambiguously determined in nine spawns from a population consisting of broodstock derived from only four families. A third experiment demonstrated that all but one of 374 individuals from 10 full-sibling families could be assigned to a family after coculture in an earthen pond for 1 year. The standardized microsatellite panels enable the development of pedigreed catfish populations and large-scale performance evaluations in common environments to support the genetic improvement of cultured catfish through selective breeding.

Genome sequence of Edwardsiella ictaluri 93-146, a strain associated with a natural channel catfish outbreak of enteric septicemia of catfish.

Edwardsiella ictaluri is the cause of extensive mortalities and economic losses to the channel catfish industry of the southeast United States. Here we report the complete genome of Edwardsiella ictaluri 93-146. Whole-genome sequence analysis of E. ictaluri provides a tool for understanding the genomic regions specific to the species and the Edwardsiella genus.

Effects of fasting on IGF-I, IGF-II, and IGF-binding protein mRNA concentrations in channel catfish (Ictalurus punctatus).

The effects of fasting on insulin-like growth factor (IGF)-I, IGF-II, and IGF-binding protein (IGFBPs) mRNA in channel catfish were examined. Fed control fish (Fed) were compared to fish that had been fasted for 30 d followed by 15 d of additional feeding (Restricted). Sequence alignment and similarity to orthologous proteins in other vertebrates provided structural evidence that the 3 catfish sequences identified in the present research were IGFBP-1, -2, and -3. Prolonged fasting (30 d) reduced body weight approximately 60% (P<0.001) and decreased IGF-I mRNA in the liver and muscle (P<0.01). Fifteen days of re-feeding restored concentrations of hepatic and muscle IGF-I mRNA. Liver IGF-II mRNA was not affected by fasting but was increased 2.2-fold after 15 d of re-feeding (P<0.05). Abundance of muscle IGF-II mRNA was similar between the fed control group and the restricted group throughout the experimental period. Fasting also increased liver IGFBP-1 mRNA (P<0.05) and decreased IGFBP-3 mRNA (P<0.01), whereas abundance of IGFBP-2 mRNA was not significantly affected. Interestingly, re-feeding for 15 d did not restore concentrations of IGFBP-1 and IGFBP-3 mRNA relative to fed control concentrations. The IGF results suggest that IGF-I and IGF-II are differently regulated by nutritional status and probably have a differential effect in promoting muscle growth during recovery from fasting. Similar to mammals, IGFBP-1 mRNA in catfish is increased during catabolism, whereas IGFBP-3 mRNA is decreased during inhibited somatic growth. The IGFBP results provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in catfish.

Expression profiles of cloned channel catfish (Ictalurus punctatus) lymphoid cell lines and mixed lymphocyte cultures.

Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLCs) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLCs have been biologically and phenotypically characterized using a variety of techniques including reverse transcription polymerase chain reaction (RT-PCR), as well as Northern and Southern blotting. To expand the molecular characterization of these cultures, microarray analysis was employed. Clonal B (3B11), macrophage (42TA), and cytotoxic T cell (TS32.15 and TS32.17) lines and MLCs were examined using a cDNA array containing approximately 2500 probes derived from EST libraries prepared from the 42TA macrophage cell line, a MLC, and 5-14-day-old catfish fry. Analysis showed that each cell line displayed a unique RNA expression profile that included a variety of immune-related genes. Pearson correlation analysis indicated that one cytotoxic T cell line (TS32.15) clustered with the MLC, whereas a second cytotoxic T cell line (TS32.17) was more closely associated with a second cluster containing B cells and macrophages. This study illustrates the utility of microarray analyses in profiling RNA expression patterns in catfish lymphoid cell lines and will serve as a platform for examining catfish immune responses following virus infection or poly [I:C] treatment.

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri.

AIMS: To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons. METHODS AND RESULTS: The Edw. ictaluri rrn operons were identified from a 5-7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I-CeuI enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri. CONCLUSIONS: The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family. SIGNIFICANCE AND IMPACT OF THE STUDY: This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.

Definition of the USDA103 strain of channel catfish (Ictalurus punctatus).

Although there are differences in performance between genetic groups of channel catfish, identification and management of these groups is difficult because catfish strains look alike and individuals cannot be tagged efficiently. Thus, US catfish producers have not been able to objectively identify fish from different strains or populations, and it has been difficult for them to maintain the genetic purity of populations on the farm. We have developed a multiplexed microsatellite genotyping system to define catfish populations based on allelic frequency and exclusion. A commercial catfish genotype database was developed using catfish samples collected from 24 processing plants in the four main US catfish-producing states. The utility of the system was tested by the molecular characterization of the USDA103 research strain. Using eight microsatellite loci, the probability of falsely classifying an individual non-USDA103 catfish as a USDA103 was 0.0065. From a sample of 50 fish from a putative USDA103 pond, the probability of falsely including two non-USDA103 fish was 1 x 10(-105), and the conservative probability of falsely excluding two USDA103 fish was 1 x 10(-6). This genotyping system provides channel catfish producers with an objective mechanism for identification and management of genetically selected fish.

Poly I:C inhibits the expression of channel catfish virus immediate-early gene ORF 1 at early times after infection.

Channel catfish virus (CCV) is a herpesvirus that infects channel catfish fry and fingerlings. Previous research has demonstrated that Type I interferons inhibit the expression of immediate-early (IE) genes of some mammalian herpesviruses. However, CCV is distantly related to the mammalian herpesviruses and Type I interferons from higher vertebrates exhibit only 20% similarity to fish interferons. In this work we demonstrate that treatment of channel catfish ovary (CCO) cells, a fibroblast-like cell line, with poly I:C, a known inducer of Type I interferons, results in inhibition of expression of the CCV IE gene ORF 1. Thus, although the genes involved have diverged, the mechanism appears to be conserved. If this paradigm holds true for other CCV IE-Type I interferon interactions, it could have important implications for the impact of CCV on the host immune system.

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene.

The catfish IGH locus is large ( approximately 1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3' end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3' C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.

Assignment of immune-related genes to the channel catfish, Ictalurus punctatus, genetic map.

Eighteen new genes, adenosine A1 receptor (ADORA1), complement component 4-beta (C4b), complement component 8-beta (C8b), chemokine ligand 19 (CCL19), chemokine ligand 21 (CCL21), chemokine ligand 25 (CCL25), chemokine receptor 2 (CCR2), chemokine receptor 5 (CCR5), chemokine receptor 4 (CCR4), chemokine receptor 7 (CCR7), chemokine receptor 9 (CCR9), interleukin 1-beta (IL1B), integrin II-beta (ITGB2), novel immune type receptor 2 (NITR2), novel immune type receptor 4 (NITR4), natural killer cell lysin (NKLYSIN), nucleotide excision repair (RAD23B) and tumour necrosis factor-alpha (TNF), were assigned to the channel catfish (Ictalurus punctatus) genetic linkage map. Polymorphic microsatellite markers were developed for NITR2, NITR4 and RAD23B from short-tandem repeats in the available sequence. Polymorphic microsatellite markers were developed for the remaining 15 genes by short-tandem repeat-anchored primer sequencing of catfish bacterial artificial chromosomes. Two gene clusters (MYOG-NRAMP-ADORA1) and (CCR4-CCR2-CCR5) displayed conservation of synteny between catfish and mammals. Assignment of 18 new genes to the catfish linkage map will further advance integration of genetic and physical maps and comparative mapping between channel catfish and map rich species.